Targeting vector-phospholipid conjugates

ABSTRACT

Peptide vectors having high KDR binding affinity and processes for making such vectors are provided. The peptide vectors may be conjugated to phospholipids and included in ultrasound contrast agent compositions. Such ultrasound contrast agents are particularly useful in therapeutic and diagnostic methods, such as in imaging KDR-containing tissue and in the evaluation and treatment of angiogenic processes associated with neoplastic conditions. The present invention also provides processes for the large scale production of highly pure dimeric and monomeric peptide phospholipid conjugates as well as precursor materials used to form the conjugates. The present invention further provides processes for the large scale production of highly pure peptide phospholipid conjugates which contain very low levels of TFA.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11,608,395, filed Dec. 8, 2006 which claims priority to and benefit ofU.S. Provisional Application No. 60/833,342, filed Jul. 25, 2006 andU.S. Provisional Application No. 60/749,240, filed Dec. 9, 2005, and isa continuation-in-part of U.S. application Ser. No. 10/661,156, filedSep. 11, 2003, which is a continuation-in-part of U.S. application Ser.No. 10/382,082, filed Mar. 3, 2003, and a continuation in-part ofInternational Application No. PCT/US03/06731, filed Mar. 3, 2003, bothof which claim priority to and benefit of U.S. Provisional ApplicationNo. 60/440,411, filed Jan. 15, 2003; and U.S. Provisional ApplicationNo. 60/360,851, filed Mar. 1, 2002, the contents of each which arehereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to targeting vector-phospholipidconjugates and particularly targeting peptide-phospholipid conjugates,which are useful in therapeutic and diagnostic compositions and methodsof preparation of the same. The invention includes targeted ultrasoundcontrast agents, and particularly targeted microbubbles which includesuch targeting vector-phospholipid conjugates.

BACKGROUND OF THE INVENTION

Angiogenesis, the formation of new blood vessels, occurs not only duringembryonic development and normal tissue growth and repair, but is alsoinvolved in the female reproductive cycle, establishment and maintenanceof pregnancy, and repair of wounds and fractures. In addition toangiogenesis that occurs in the normal individual, angiogenic events areinvolved in a number of pathological processes, notably tumor growth andmetastasis, and other conditions in which blood vessel proliferation isincreased, such as diabetic retinopathy, psoriasis and arthropathies. Inaddition, angiogenesis is important in the transition of a tumor fromhyperplastic to neoplastic growth. Consequently, inhibition ofangiogenesis has become an active cancer therapy research field.

Tumor-induced angiogenesis is thought to depend on the production ofpro-angiogenic growth factors by the tumor cells, which overcome otherforces that tend to keep existing vessels quiescent and stable. The bestcharacterized of these pro-angiogenic agents or growth factors isvascular endothelial growth factor (VEGF) (Cohen et al., FASEB J., 13:9-22 (1999)). VEGF is produced naturally by a variety of cell types inresponse to hypoxia and some other stimuli. Many tumors also producelarge amounts of VEGF, and/or induce nearby stromal cells to make VEGF(Fukumura et al., Cell, 94: 715-725 (1998)). VEGF, also referred to asVEGF-A, is synthesized as five different splice isoforms of 121, 145,165, 189, and 206 amino acids. VEGF₁₂₁ and VEGF₁₆₅ are the main formsproduced, particularly in tumors (see Cohen et al. 1999, supra). VEGF₁₂₁lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and doesnot bind to heparin or extracellular matrix, unlike VEGF₁₆₅. Each of thereferences cited in this paragraph is incorporated by reference in itsentirety.

VEGF family members act primarily by binding to receptor tyrosinekinases. In general, receptor tyrosine kinases are glycoproteins havingan extracellular domain capable of binding one or more specific growthfactors, a transmembrane domain (usually an alpha helix), ajuxtamembrane domain (where the receptor may be regulated, e.g., byphosphorylation), a tyrosine kinase domain (the catalytic component ofthe receptor), and a carboxy-terminal tail, which in many receptors isinvolved in recognition and binding of the substrates for the tyrosinekinase. There are three endothelial cell-specific receptor tyrosinekinases known to bind VEGF: VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), andVEGFR-3 (Flt4). Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which areused interchangeably herein) have been identified as the primary highaffinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDRdisplays more abundant endothelial cell expression (Bikfalvi et al., J.Cell. Physiol., 149: 50-59 (1991)). Moreover, KDR is thought to dominatethe angiogenic response and is therefore of greater therapeutic anddiagnostic interest (see Cohen et al. 1999, supra). Expression of KDR ishighly upregulated in angiogenic vessels, especially in tumors thatinduce a strong angiogenic response (Veikkola et al., Cancer Res., 60:203-212 (2000)). The critical role of KDR in angiogenesis is highlightedby the complete lack of vascular development in homozygous KDR knockoutmouse embryos (Folkman et al., Cancer Medicine, 5th Edition (B. C.Decker Inc.; Ontario, Canada, 2000) pp. 132-152).

KDR (kinase domain region) is made up of 1336 amino acids in its matureform. The glycosylated form of KDR migrates on an SDS-PAGE gel with anapparent molecular weight of about 205 kDa. KDR contains sevenimmunoglobulin-like domains in its extracellular domain, of which thefirst three are the most important in VEGF binding (Cohen et al. 1999,supra). VEGF itself is a homodimer capable of binding to two KDRmolecules simultaneously. The result is that two KDR molecules becomedimerized upon binding and autophosphorylate, becoming much more active.The increased kinase activity in turn initiates a signaling pathway thatmediates the KDR-specific biological effects of VEGF.

Thus, not only is the VEGF binding activity of KDR in vivo critical toangiogenesis, but the ability to detect KDR upregulation on endothelialcells or to detect VEGF/KDR binding complexes would be extremelybeneficial in detecting or monitoring angiogenesis.

It is well known that gas filled ultrasound contrast agents areexceptionally efficient ultrasound reflectors for echography. Suchultrasound contrast agents include, for example, gas-filledmicrovesicles such as gas-filled microbubbles and gas filledmicroballoons. Gas filled microbubbles are particularly preferredultrasound contrast agents. (In this disclosure the term of“microbubble” specifically designates a gaseous bubble surrounded orstabilized by phospholipids). For instance injecting into thebloodstream of living bodies suspensions of air- or gas-filledmicrobubbles in a carrier liquid will strongly reinforce ultrasonicechography imaging, thus aiding in the visualization of internalanatomical structures. Imaging of vessels and internal organs canstrongly help in medical diagnosis, for instance for the detection ofneoplastic, cardiovascular and other diseases.

For both diagnostic and therapeutic purposes it would be particularlybeneficial to incorporate into gas filled ultrasound contrast agents,targeting vector compositions which exhibit high binding affinity for adesired target (such as, for example, KDR or the VEGF/KDR complex). Forexample, targeting vector—phospholipid conjugates and particularlytargeting peptide-phospholipid conjugates may be used to preparetargeted, gas filled ultrasound contrast agents. In addition, it wouldbe particularly beneficial to have methods for large scale production ofhighly purified forms of such targeting vector—phospholipid conjugates.Such compositions and methods would allow for production of compositionsfor use in diagnostic or therapeutic applications such as, for example,precise targeting of reporter moieties, tumoricidal agents orangiogenesis inhibitors to the target site.

SUMMARY OF THE INVENTION

The present invention provides targeting vector-phospholipid conjugatesand particularly targeting peptide-phospholipid conjugates which areuseful in the preparation of gas filled ultrasound contrast agents. In apreferred embodiment the targeting peptide-phospholipid conjugatesinclude targeting peptides which exhibit high KDR binding affinity andthus are useful components of contrast agents for imaging ofangiogenesis processes.

The present invention also provides monomeric and dimeric peptidephospholipid conjugates (also referred to herein as lipopeptides) whichare useful in preparing gas filled ultrasound contrast agents, andparticularly in preparing ultrasound contrast agents which target KDRand may be used for imaging of angiogenesis processes.

The present invention also provides methods and processes for the largescale production of highly pure monomeric and dimeric peptidephospholipid conjugates, particularly monomeric and dimeric peptidephospholipids conjugates having high KDR binding affinity.

The present invention also provides methods and processes for the largescale production of highly pure dimeric peptide phospholipid conjugateshaving minimal levels of trifluoroacetic acid (TFA).

The present invention also provides methods for synthesizing monomericpeptides in high purity and the construction of peptide phospholipidconjugates from multiple peptide sub-units.

The present invention also provides monomeric peptides which bind KDR orthe VEGF/KDR complex with high affinity, as well as methods ofsynthesizing and using such monomeric peptides.

The present invention also provides targeted ultrasound contrast agentsprepared from such targeting vector-phospholipid conjugates. Suchtargeted ultrasound contrast agents are useful for imagingtarget-bearing tissue. In a preferred embodiment, the targetedultrasound contrast agents are targeted microbubbles and the targetingvector-phospholipid conjugates include targeting peptides which exhibithigh KDR binding affinity and thus are useful components of contrastagents for imaging KDR-bearing tissue and particularly for imaging oftumors and angiogenesis processes. Methods of preparing and using suchtargeted ultrasound contrast agents are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method for the production of a monomeric peptidephospholipid conjugate (1) (SEQ ID NO: 1) from a linear peptide monomer(2) (SEQ ID NO: 2), via intermediate (3) (SEQ ID NO: 3).

FIG. 2 illustrates a monomeric peptide phospholipid conjugate (1)including a peptide with high binding affinity for KDR.

FIG. 3 illustrates a method for the production of a precursor dimerpeptide (16) from peptide monomers.

FIG. 4 illustrates a method for the conjugation of the precursor dimerpeptide shown in FIG. 1 to DSPE-PEG2000-NH₂ to form a dimeric peptidephospholipid conjugate (11) containing peptides which bind with highaffinity to KDR.

FIG. 5 illustrates a dimeric peptide-phospholipid conjugate (11)containing peptides which bind with high affinity to KDR.

FIG. 6 illustrates a method for the production of dimerpeptide-phospholipid conjugates (such as (21)) having minimal levels ofTFA.

FIG. 7 illustrates another method for the production of dimerpeptide-phospholipid conjugates (such as (21)) having minimal levels ofTFA.

FIG. 8 illustrates another method for the production of dimerpeptide-phospholipid conjugates having minimal levels of TFA.

FIG. 9 illustrates another representative monomeric peptide (32) havinga high binding affinity for KDR.

FIG. 10 illustrates another monomeric peptide-phospholipid conjugate(31) which includes the monomeric peptide shown in FIG. 9.

FIGS. 11A-C show images obtained by using the dimer peptide-phospholipidconjugate (11) (shown in FIG. 52) in a contrast agent at: 1) baseline(FIG. 11A); 2) after 25 minutes (FIG. 11B); and 3) after subtraction ofthe baseline and free circulating bubbles (FIG. 11C).

FIGS. 12A-C show images obtained by using the monomeric phospholipidpeptide conjugate (1) (shown in FIG. 2) in a contrast agent at baseline(FIG. 12A); after 25 minutes (FIG. 12B); and after subtraction of thebaseline and free circulating bubbles (FIG. 12C).

DETAILED DESCRIPTION

Applicants have unexpectedly discovered peptide phospholipid conjugates,which are useful in producing targeted ultrasound contrast agents andwhich have exceptional KDR binding efficiency. These compounds includemonomeric peptide phospholipid conjugates having a linear peptidemonomer which binds with high affinity to KDR and dimeric peptidephospholipid conjugates having two distinct monomer subunits, eachbinding to KDR. In addition, highly efficient methods for large scaleproduction of purified forms of these conjugates and precursor materialshave been discovered. Such methods include the production of dimericpeptide phospholipid conjugates having minimal levels of TFA.

The phospholipid may be selected from the group consisting of:phosphatidylethanolamines and modified phosphatidylethanolamines.Particularly preferred phospholipids include phosphatidylethanolaminesmodified by linking a hydrophilic polymer thereto. Examples of modifiedphosphatidylethanolamines are phosphatidylethanolamines (PE) modifiedwith polyethylenglycol (PEG), in brief “PE-PEGs”, i.e.phosphatidylethanolamines where the hydrophilic ethanolamine moiety islinked to a PEG molecule of variable molecular weight (e.g. from 300 to5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG.DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred,with DSPE-PEG2000 particularly preferred. Note that a salt form of thephospholipid may be used, such as, for example, the trimethyl ammoniumsalt, the tetramethylammonium salt, the triethylammonium salt, sodiumsalt, etc.

These compounds may be incorporated into gas filled ultrasound contrastagents, such as, for example, gas filled microbubbles to form contrastagents that provide excellent imaging of target-bearing tissue. In apreferred embodiment, targeting vector-phospholipid conjugates whichinclude targeting peptides which bind with high affinity to KDR areincorporated into targeted microbubbles. As shown herein, such targetedmicrobubbles selectively localize at KDR-bearing tissue, permittingimaging of such tissue, and, in particular imaging of tumors andangiogenic processes, including those processes associated withneoplastic development.

Monomer Conjugates

Generally

Table 1 provides a description for the identification labels shown inFIGS. 1, 2, 9 and 10.

TABLE 1 1 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000- NH-Glut)-NH₂(SEQ ID NO. 1) 2 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO. 2) 3mono-NHS ester of glutaryl-peptide monomer (2)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS-Glut)-NH₂ (SEQ ID NO. 3) 4DSPE-PEG2000-NH₂ phospholipid1,2-distearoyl-sn-glycero-3-phosphoethanol-aminocarbonyloxy-(PEG2000)-amine 31 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000- NH-Glut)-NH₂(SEQ ID NO. 4) 32  Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO. 5)

As shown if FIGS. 1 and 2 the monomeric peptide phospholipid conjugate(1)N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-Ll-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysinamide,is a phospholipid conjugate. This conjugate is also referred to asAc-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ IDNO. 1) andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH₂.It comprises a 29 amino acid linear peptide monomer (2)N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-Ll-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide,also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO. 2)andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH₂.This novel peptide monomer binds with high affinity to KDR. It should beunderstood that analogs and derivatives of the monomeric peptidephospholipid conjugate (1) and the linear peptide monomer (2) areintended to be included within the scope of the present invention.

FIG. 10 provides the structure of another monomeric peptide phospholipidconjugate (31),N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysine-amide,a phospholipid conjugate. This conjugate is also referred to asAc-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ ID NO. 4)andAc-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH₂.As shown in FIG. 9, the conjugate comprises a 28 amino acid linearpeptide monomer (32),N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide,which is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ IDNO. 5) andAc-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH₂.As shown in co-pending application, U.S. application Ser. No.10/661,156, filed Sep. 11, 2003, this peptide monomer binds with highaffinity to KDR. It should be understood that analogs and derivatives ofthe monomeric peptide phospholipid conjugate and the linear peptidemonomer are intended to be included within the scope of the presentinvention.

Additional monomeric peptide phospholip conjugates according to thepresent invention include Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. IDNO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7). Thesepeptide monomers bind with high affinity to KDR. It should be understoodthat analogs and derivatives of these monomeric peptide monomers andpeptide phospholipids conjugates comprising these monomers are intendedto be included within the scope of the present invention.

As shown in the Examples, ultrasound contrast agents such as gas filledmicrobubbles formulated with the monomeric peptide phospholipidconjugates (1) and (31) displayed high KDR binding which was confirmedusing echographic examination of VX2 tumors in rabbits.

Methods of Preparation of Monomer Peptide-Phospholipid Conjugates

Ideally, to facilitate production of a monomeric peptide phospholipidconjugate, the linear peptide monomer should be prepared in bulk. Thenconjugation of the purified linear peptide monomer to the phospholipid,such as, for example, a pegylated phospholipid in salt form, e.g.,DSPE-PEG2000-NH₂ phospholipid ammonium salt via the linkerdisuccinimidyl glutarate (DSG), may be used to provide monomeric peptidephospholipid conjugates.

In preparing monomeric peptide phospholipid conjugates, methodsaccording to the present invention provide at least the followingadvantages: increased yield of peptide synthesis; reduced extent ofracemization; avoidance of previously observed piperidine amideformation during synthesis, efficient purification of peptide monomers,development of a procedure for conjugation of peptide monomers on largerscale; and development of purification protocols that would allow theready separation of the monomeric peptide phospholipid conjugates fromthe starting DSPE-PEG2000-NH₂ phospholipid ammonium salt.

With respect to the methods described below (including the Examplessection of this specification), it should be understood that while suchmethods may specifically reference the use of linear peptide monomers(2) and (32) to form monomeric peptide phospholipids conjugates (1) and(31), these methods also may be used with peptide monomersAc-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6) andAc-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7), as well as analogsand derivatives of any of the above. It also should be understood thatthe numerical values referred to in the synthesis of monomeric peptidephospholipid conjugates are representative.

Linear peptide monomers may be prepared by SPPS. The sequence of thelinear peptide monomers may be constructed as a C-terminal carboxamideon Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Peptide synthesismay be accomplished using Fmoc chemistry on a SONATA®/Pilot PeptideSynthesizer. Problems previously observed with this process have beenracemization, incomplete couplings and piperidine amide formation, eachof which contribute to suboptimal yield and purity. A dramatic decreasein the formation of the piperidine amide may be attained by the use of25% piperidine in DMF containing HOBt (0.1M) as the reagent for Fmocremoval. Racemization may be considerably reduced by using DIC/HOBt asthe activator for most couplings; a 3 h coupling time using a four-foldexcess of pre-activated Fmoc-amino acid with an intervening wash withanhydrous DMF (6×). N′-Fmoc amino acids may be dissolved just beforetheir coupling turn and pre-activated with DIC/HOBt in DMF for 4 min andtransferred to the reaction vessel. This may be accomplished on theSonata instrument by loading the solid Fmoc-amino acids into the aminoacid vessels of the instrument and then programming the instrument toadd DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of thesolution.

To optimize the yield, the problem of aggregation of the resin duringthe synthesis of longer peptides, which can be devastating even whenoptimal coupling reagents are employed, may be addressed. To reduceaggregation during peptide assembly the strategy of using pseudoprolinedipeptides to incorporate X-Thr or X-Ser as dipeptides instead ofsequential couplings of X and Thr or X and Ser, may be employed. Forlinear peptide monomers sequential couplings of Leu¹¹-Ser andLeu²²-Ser²³ may be replaced by the single coupling of the pseudoprolinedipeptide, Fmoc-Leu-Ser(ψ^(Me,Me)pro)-OH. Additional optimization may beaccomplished by reducing the number of couplings by usingFmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling ofFmoc-Gly-OH. Activation of -Gly-Gly-OH segments may lead to cyclizationof the activated acid function with the distal amide function to producean inactive diketopiperazine; this may reduce coupling yields in a timedependant manner. This problem may be avoided by addition ofFmoc-Gly_(n)-OH (n=2, 3) to the reaction vessel and sequential additionof HOBt and DIC; the activated Fmoc-Gly_(n)-OH may be intercepted by theresin-bound amino group before appreciable cyclization to thediketopiperazine takes place. With these improvements, the synthesis oflinear peptide monomers may be completed on the Sonata PeptideSynthesizer on a 10 mmol synthesis scale.

After chain elongation, the Fmoc may be removed from the N-terminus. Thepeptide and the free amino group may be acetylated. Then the peptidesequence may be cleaved from the resin and deprotected using “Reagent B”(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. Afterthe cleavage reaction the crude peptide may be isolated as a solid byevaporation of the volatiles, trituration of the residue with diethylether and washing of the solid thus obtained using the same solvent. Inanother variation the peptide may be precipitated from the reactionmixture by addition of diethyl ether to the reaction mixture, collectingthe solid thus formed and washing with the same solvent.

Linear peptide monomers may be purified as described below. Again, thenumerical references are representative. Crude linear peptide monomers(0.5 g) may be dissolved in CH₃CN (40 mL/g) and this solution may bediluted to a final volume of 100 mL with water. The solution may then befiltered. The filtered solution may be loaded onto the preparative HPLCcolumn (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibratedwith 10% CH₃CN in water (0.1% TFA). After loading, the composition ofthe eluent may then be ramped to 20% CH₃CN-water (0.1% TFA) over 1 min,and a linear gradient may be initiated at a rate of 0.6%/min of CH₃CN(0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractionsmay be checked for purity on an analytical reversed phase C18 column(Waters XTerra MS-C18, 10μ, 120 Å, 4.6×50 mm) and fractions containingthe product in >95% purity may be combined and freeze-dried. For eachpurification of 0.5 g of crude peptide 0.12 g (24%) of linear peptidemonomer may be consistently isolated and will provide the peptide in thesame yield and purity.

Synthesis of monomeric peptide phospholipid conjugates may be performedas described below. The numerical references are again representative.The last step in the synthesis may be the conjugation of thephospholipid, such as, for example, a pegylated phospholipid such asDSPE-PEG2000-NH₂ phospholipid ammonium salt to a linear peptide monomer.The PEG2000 moiety of DSPE-PEG2000-NH₂ phospholipid ammonium salt (4) isnominally comprised of 45 ethylene glycol units. It should beunderstood, however, that this material is a distribution of PEGcontaining species whose centroid is the nominal compound containing 45ethylenoxy units. The conjugation of a linear peptide monomer withDSPE-PEG2000-NH₂ phospholipid ammonium salt may be accomplished bypreparation of the glutaric acid monoamide mono NHS ester of a linearpeptide monomer and reaction of this with the free amino group of thephospholipid ammonium salt. Thus a linear peptide monomer may be reactedwith DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. Thereaction mixture may be diluted with ethyl acetate, which may result inprecipitation of the peptide glutaric acid monoamide mono-NHS ester. Thesupernatant containing un-reacted DSG may be decanted and theintermediate peptide mono-NHS ester may be washed several times withethyl acetate to remove traces of DSG. Mass spectral data confirms theformation of the peptide mono-NHS ester as a clean product. The solidmono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2000-NH₂phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for24 h. The linear peptide monomer glutaric acid monoamide mono-NHS estermay be used in excess to maximize the consumption of the phospholipidammonium salt because free phospholipid ammonium salt may complicate theisolation of monomeric peptide phospholipid conjugates in highly pureform.

The reaction mixture may be diluted with a 1:1 mixture of water (0.1%TFA) and CH₃CN—CH₃OH (1:1, v/v) (0.1% TFA) (˜100 mL), applied to areversed phase C2 column (Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm, flowrate 100 mL/min) and the column may be eluted with a 3:1 mixture ofwater (0.1% TFA) and CH₃CN—CH₃OH (1:1, v/v) (0.1% TFA) to removehydrophilic impurities. Then the product may be eluted using a gradientof CH₃CN—CH₃OH (1:1) (0.1% TFA) into water (0.1% TFA) (see ExperimentalSection for details). The collected fractions may be analyzed byreversed phase HPLC using an ELS detector which allows the detection ofthe desired product and the often difficult-to-separate DSPE-PEG2000-NH₂phospholipid which has very little UV absorbance. This indicates theclear separation of the monomeric peptide phospholipid conjugates andDSPE-PEG2000-NH₂ phospholipid. The pure product-containing fractions maybe collected, concentrated on a rotary evaporator (to reduce the contentof methanol) and freeze-dried to provide monomeric peptide phospholipidconjugates as a colorless solid. In order to prepare the requiredquantity of the monomeric peptide phospholipid conjugates, several runsmay be conducted employing 0.5 g to 1.0 g of linear peptide monomer. Inall cases the target monomeric peptide phospholipid conjugates may bewere isolated in high yield and purity (e.g., 57-60% yield and >99%purity).

Dimer Conjugate

Generally

Table 2 provides a description for the identification labels shown inFIGS. 3, 4 and 5.

TABLE 2 11 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWLDSWGGLVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂ cyclic (2-12)disulfide}-NH₂ cyclic (6-13) disulfide 12Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (6-13) disulfide 13Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide 14mono-NHS ester of glutaryl-peptide 12Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH₂ cyclic (6-13) disulfide 15ivDde-bearing dimer Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(ivDde)]-NH₂ cyclic (2-12)disulfide}-NH₂ cyclic (6-13) disulfide 16Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH₂ cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide 17 Mono-NHS ester of glutaryl-peptide 16Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(NHS-Glut)]-NH₂ cyclic (2-12)disulfide}-NH₂ cyclic (6-13) disulfide 18 DSPE-PEG2000-NH₂ phospholipid

As shown in those figures the dimeric peptide phospholipid conjugate(11)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-l-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide, consistsof two monomeric peptide chains which bind KDR: a 21 amino acid cyclicdisulfide peptide monomer (13)Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amidecyclic (2-12) disulfide, and a 22 amino acid cyclic disulfide peptidemonomer (12)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysinamidecyclic 6-13 disulfide tethered by a glutaryl linker. It should beunderstood that analogs and derivatives of the dimeric peptidephospholipid conjugate (11) and the cyclic disulfide peptide monomers(12) and (13) are intended to be included within the scope of thepresent invention.

Ultrasound contrast agents (e.g. gas filled microbubbles) formulatedwith the dimeric peptide phospholipid conjugate (11) displayed high KDRbinding which was confirmed using echographic examination of VX2 tumorsin rabbits.

Methods of Preparation of Dimer-Phospholipid Conjugates

To accomplish synthesis of the dimeric peptide phospholipid conjugate(11), the monomers used for this purpose optimally should be prepared inbulk. Then the monomers may be tethered to each other usingdi-succinimidyl glutarate as a linker to form the precursor dimerpeptide (16),Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide. Thenconjugation of the purified precursor dimer peptide (16) to aDSPE-PEG2000-NH₂ phospholipid ammonium salt (18) again viadisuccinimidyl glutarate may be used in order to provide the targetdimeric peptide phospholipid conjugate (11).

In preparing dimeric peptide phospholipid conjugate (11), methodsaccording to the present invention provide at least the followingadvantages: increased yield of automated chain elongation of the peptidesequences; reduced extent of racemization encountered during synthesis;avoidance of previously observed piperidine amide formation duringsynthesis of peptide monomer (13); cyclization of linear di-cysteinecontaining peptide precursors of (12) and (13) using procedures amenableto multigram scale yet allowing efficient and practical sample handling;efficient purification of monomer peptides (12) and (13); maximizedyield and purity of precursor dimer peptide (16); development of aprocedure for conjugation of the precursor dimer peptide (16) on largerscale; and development of purification protocols that would allow theready separation of the target dimeric peptide phospholipid conjugate(11) from phospholipid ammonium salt (18).

The dimeric peptide phospholipid conjugate (11) may be prepared byautomated synthesis of the peptide monomers (12),Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH₂cyclic (6-13) disulfide, and (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂cyclic (2-12) disulfide, their efficient coupling using disuccinimidylglutarate (DSG) to give an ivDde-protected dimer, its deprotection andsubsequent coupling to DSPE-PEG2000-NH₂, also via a glutaryl linkage.Using procedures according to the present invention, monomer peptidesmay be synthesized on a 10 mmol scale without complication and afterHPLC purification may be obtained in about 20% yield and >95% purity.Such methods allow dimer formation reactions and the subsequentconjugation to the phospholipid component providing formation of dimericpeptide phospholipid conjugate (11) to be carried out on a gram scale.The precursor dimer peptide (16) may be obtained from the monomerpeptides routinely in about 32% yield and >95% purity. The dimericpeptide phospholipid conjugate (11) may be produced from the precursordimer peptide (16) in 57-60% yield and >99% purity.

Dimeric peptide phospholipid conjugates may be prepared as describedbelow. It should be appreciated that the numerical values referred to inthis representative description of the synthesis of dimeric peptidephospholipid conjugates are representative.

Described below is a representative method for the solid phase synthesisand disulfide cyclization of a peptide monomer (12)Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH₂cyclic (6-13) disulfide, and a peptide monomer (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂cyclic (2-12) disulfide.

The peptides may be constructed as their C-terminal carboxamides onPal-Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation maybe accomplished using Fmoc chemistry employing optimized deprotectionand coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10mmol synthesis scale. The optimized synthesis of the peptides byautomated SPSS may be developed by study of peptide impurities and theeffect of changes of particular elements of the protocols on the overallyield and purity of the peptides obtained.

Analysis of the impurities obtained from nonoptimized syntheses of themonomer peptides indicates that the major problems are racemization,incomplete couplings and piperidine amide formation (presumably via anintermediate aspartimide or glutarimide intermediate), each of whichcontributes to suboptimal yield and purity. A dramatic decrease information of the piperidine amide may be attained by the use of 25%piperidine in DMF containing HOBt (0.1M) as the reagent for fmocremoval. Racemization may be considerably reduced by using DIC/HOBt asthe activator for most couplings; and a 3 h coupling time using afour-fold excess of pre-activated Fmoc-amino acid with an interveningwash with anhydrous DMF (6×). N-^({tilde over (α)})Fmoc amino acids maybe dissolved just before their coupling turn and pre-activated withDIC/HOBt in DMF for 4 min and transferred to the reaction vessel. Thismay be accomplished on the Sonata instrument by loading the solidFmoc-amino acids into the amino acid vessels of the instrument and thenprogramming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentiallywith bubbling of the solution after each addition.

To optimize the yield, the problem of aggregation of the resin duringthe synthesis of longer peptides, which can be devastating even whenoptimal coupling reagents are employed, may be addressed. To reduceaggregation during peptide assembly the strategy of using pseudoprolinedipeptides to incorporate X-Thr or X-Ser (X refers to the n−1 amino acidof the sequence) as dipeptides instead of sequential couplings of X andThr or X and Ser, may be employed. Thus, for the monomer (12),Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH₂cyclic (6-13) disulfide, sequential coupling of suitably protected Thrand Gly (shown in bold above) may be replaced by the single coupling ofthe pseudoproline dipeptide, Fmoc-Gly-Thr(ψ^(Me,Me)pro)-OH. Similarly,in the synthesis of the monomer (13),Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂cyclic (2-12) disulfide, the pseudoproline dipeptide,Fmoc-Asp(OtBu)-Ser(ψ^(Me,Me)pro)-OH may be employed to replace thesequential coupling of suitably protected Ser and Asp (shown in boldfont above). Further optimization may be accomplished by reducing thenumber of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, inlieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OHsegments can lead to cyclization of the activated acid function with thedistal amide function to produce an inactive diketopiperazine; this mayreduce coupling yields in a time dependant manner. This problem may beavoided by addition of Fmoc-Gly_(n)-OH (n=2, 3) to the reaction vesseland sequential addition of HOBt and DIC; the activated Fmoc-Gly_(n)-OHmay be intercepted by the resin-bound amino group before appreciablecyclization to the diketopiperazine takes place. After chain elongationis completed the N-terminal Fmoc protecting group may be removed fromeach of the peptides and the free amino group may be acetylated.

The pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may beused to enable the selective unmasking of the ε-amine of the C-terminallysine of the monomer and dimer peptides and their subsequentfunctionalization, which also may be optimized. The ivDde group on theε-amine of the C-terminal lysine of each of the peptide monomers may beremoved using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) orLys(ivDde) for monomer (12) may be appended to the exposed lysineε-amino group using 4 equivalents of the Fmoc amino acid and 4equivalents each of DIC and HOBt in DMF for 10 h. After completion ofthe synthesis, the peptide sequence may be cleaved from the resin anddeprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane,88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction was complete thepeptide may be precipitated, washed with diethyl ether and dried.

The following procedures for cyclization of the linear di-cysteinecontaining peptides may be used to provide optimal scale-up of monomerpeptides. Generally the aerial oxidation of linear di-cysteine peptidesmay be carried out at ca 0.5-5 mg/mL (for the disclosed peptide monomers˜0.18-1.8 mM in peptide, ˜0.36-3.6 mM in cysteine thiol). In order towork at significantly higher concentrations DMSO-assisted cyclization ofdi-cysteine peptides allows the cyclization of ˜10 g of the linearpeptides in good yields in as little as ˜50 mL of solution. Thereforethe crude linear di-cysteine peptides may be cyclized in 95% DMSO-H₂O (5mL/g) at pH 8.5 at ambient temperature. The progress of the cyclizationmay be routinely followed by mass spectroscopy and HPLC. Althoughcyclization may be essentially complete in ˜36 h, the reaction mixturemay be generally stirred for up to 48 h. The cyclic disulfide peptidesmay be precipitated from the reaction mixture by dilution with CH₃CN andthe resulting off-white crude solid peptides may be collected byfiltration. This is a convenient method for removing DMSO from the crudecyclic peptide.

Purification and isolation of monomer peptide (12),Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH₂ may be accomplished asdescribed below. Note that as used herein the designation “C*” refers toa cysteine residue that contributes to a disulfide bond. Attempts todissolve 0.5 g of the crude peptide in up to 300 mL of 30% CH₃CN inwater (0.1% TFA) have been unsuccessful. Therefore, as an alternative,the crude peptide, (0.5 g) may be dissolved in DMSO (5 mL/g) and thissolution may be diluted to a final volume of 100 mL with 20%CH₃CN-water. The solution may be filtered. The filtered solution may beloaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18,10μ, 300 Å, 50×250 mm) equilibrated with 10% CH₃CN (0.1% TFA) in water(0.1% TFA), and the column may be eluted with 10% CH₃CN (0.1% TFA) inwater (0.1% TFA) to wash DMSO from the column. The composition of theeluent then may be ramped to 35% CH₃CN-water (01% TFA) over 1 min, and alinear gradient may be initiated at a rate of 0.5%/min of CH₃CN (0.1%TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may bechecked for purity on an analytical reversed phase C18 column (WatersXTerra MS-C18, 10, 120 Å, 4.6×50 mm) and fractions containing theproduct in >95% purity may be combined and freeze-dried. For eachpurification of 0.5 g of crude peptide 0.1 g (20%) for (12),Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]-NH₂ may be isolated. Repeatpurifications have been found to provide the peptide consistently in thesame yield and purity.

The peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂ maybe purified and isolated as described for peptide monomer (12) exceptthat the subject peptide may be dissolved in 20% CH₃CN (0.1% TFA) in0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containingdiluent. The resulting solution of crude peptide may be loaded onto thepreparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250mm, flow rate 100 mL/min) equilibrated with 10% CH₃CN in water (0.1%TFA). The column may be eluted with 10% CH₃CN (0.1% TFA)/water (0.1%TFA) at 100 mL/min for 5 min. Then the composition of the eluent may beramped to 30% CH₃CN (0.1% TFA)/water (0.1% TFA) over 1 min and a lineargradient rate of 0.5%/min of CH₃CN (0.1% TFA) into water (0.1% TFA) maybe initiated, and maintained until the desired peptide is completelyeluted from the column. Product-containing fractions may be analyzed ona Waters XTerra analytical reversed phase C-18 column (10μ, 120 Å) andfractions containing the product in >95% purity may be pooled andfreeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12g, 24% yield) in >95% purity. The 10 g of crude peptide monomer may bepurified serially in this manner.

Described below is a representative method for preparing the precursordimer peptide (16),Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide. The preparation ofthe precursor dimer peptide may be accomplished by the tethering of themonomer peptides in a two step procedure. First,Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH₂ (12) may be reacted withdisuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate,which results in precipitation of the glutaric acid monoamide mono-NHSester of the peptide. The supernatant, containing unreacted DSG, may bedecanted and the mono-NHS ester may be washed several times with ethylacetate to remove traces of DSG. Mass spectral data confirms theformation of the mono-NHS ester as a clean product. This may beredissolved in DMF and reacted with monomer peptideAc-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂ (13) in the presence of DIEA(5 eq). HPLC and MS results indicate the formation of the ivDde-bearingdimer, as a single major product. The ivDde protecting group on theε-amine of Lys of the dimer may be removed by stirring the reactionmixture with hydrazine (10%) in DMF for 20 min. The solution then may beacidified with TFA and diluted with 10% CH₃CN (0.1% TFA)-water (0.1%TFA), applied to a preparative reversed phase C18 HPLC column andpurified by a gradient elution of acetonitrile (0.1% TFA) into 0.1%aqueous TFA. In order to provide the needed quantity of the precursordimer peptide, the reaction may be conducted employing from 0.5 g to asmuch as 1 g of each of the monomer peptides. In every case the requiredprecursor dimer peptide may be isolated in ˜32% yield and >95% purityconfirming the reproducibility and scalability of the procedures.

The final step in the synthesis may be the conjugation ofDSPE-PEG2000-NH₂ phospholipid ammonium salt (18) to the precursor dimerpeptide. As mentioned previously, the PEG2000 moiety of DSPE-PEG2000-NH₂is nominally comprised of 45 ethylene glycol units. It should beunderstood, however, that this material is a distribution of PEGcontaining species whose centroid is the nominal compound containing 45ethylenoxy units.

Conjugation of the DSPE-PEG2000-NH₂ to the precursor dimer peptide maybe accomplished by preparation of a glutaric acid monoamide mono NHSester of the precursor dimer and reaction of this with the free aminogroup of the phospholipid ammonium salt. Thus the ivDde bearingprecursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF inthe presence of DIEA (5 eq.) for 30 min. As in the preparation of theprecursor dimer peptide the solution may be diluted with ethyl acetateto precipitate the glutaric acid monoamide mono-NHS ester of the dimer(17), as a solid. The supernatant may be decanted to remove theun-reacted DSG. The solid glutaric acid monoamide mono-NHS ester of thedimer peptide (17) may then be washed several times with ethyl acetateto remove traces of DSG. Mass spectral results confirm the formation ofthe glutaric acid monoamide mono-NHS ester of the peptide dimer as aclean product.

The dimer glutaric acid monoamide mono-NHS ester (17) may be dissolvedin DMF-CH₂Cl₂ (8:2) and reacted with DSPE-PEG2000-NH₂ phospholipidammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. TheNHS ester (17) may be used in excess to maximize the consumption of thephospholipid ammonium salt because any free phospholipid may complicatethe purification and isolation of the final product. The reactionmixture may be diluted with water (0.1% TFA)-CH₃CN—CH₃OH (1:1) (0.1%TFA) (˜1100 mL), applied to a reversed phase C4 column (Kromasil® PrepC4, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may beeluted with water (0.1% TFA)-CH₃CN—CH₃OH (1:1) (0.1% TFA) solventmixture to remove hydrophilic impurities. Then the product may be elutedusing a gradient of CH₃CN—CH₃OH (1:1) (0.1% TFA) into water (0.1% TFA).The collected fractions may be analyzed by reversed phase HPLC using anELS detector which allows the detection of the desired product and theoften difficult to separate DSPE-PEG2000-NH₂ phospholipid ammonium saltwhich has no strong UV chromophore. This indicates the clear separationof dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH₂phospholipid ammonium salt. The pure product-containing fractions may becollected, concentrated on a rotary evaporator (to reduce the content ofmethanol) and freeze-dried to provide the dimer peptide phospholipidconjugate as a colorless solid.

In order to prepare the required quantity of the dimer peptidephospholipid conjugate, several runs may be conducted employing 0.5 g to1.0 g of the precursor dimer peptide. In all cases the target dimerpeptide phospholipid conjugate may be isolated in 57-60% yield andin >99% purity. The bulk quantity of dimer peptide phospholipidconjugate, obtained from the serial runs described above may be obtainedby dissolution of the product from the individual runs int-butanol-acetonitrile-water (1:1:3) followed by lyophilization. Theprocedure of Ellman for quantitative estimation of free thiol may beapplied to the bulk sample of the dimeric peptide phospholipidconjugate; free thiol, if present will be below the limit of detection.Amino acid composition analysis gives results within the acceptablelimits, supporting the assigned structure of the peptide derivative.MALDI-TOF mass spectral analysis also supports the presumed structure ofthe dimeric peptide phospholipid conjugate.

Methods of Preparation of Dimer-Phospholipid Conjugates Having Low orNegligible Levels of TFA

The present invention also provides methods for producing dimericpeptide-phospholipid conjugates having very low levels of TFA. Whilecertain methods provide for the synthesis and purification of suchconjugates on a gram scale, formation of a lyso-version of theconjugates has been observed upon storage of lyophilized material at 5°C. or upon storage of aqueous solutions of the conjugates. It isbelieved that the lyso-compound is formed by TFA-promoted acidhydrolysis of one of the phospholipid fatty acid esters in dimerpeptide-phospholipid conjugates.

To obtain the phospholipid peptide as a stable material bearing apharmaceutically acceptable counterion, highly efficient methods forobtaining dimer peptide-phospholipid conjugates were discovered whichconvert the TFA salts of the dimer peptide-phospholipid conjugate, orany suitable precursor(s), to analogous pharmaceutical acetate salt(s).Representative embodiments of these methods are provided below.

Table 3 provides a description for the identification labels shown inFIGS. 6, 7 and 8.

TABLE 3 21 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂ cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide 22Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (2-12) disulfide nTFA 23Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH₂ cyclic (2-12) disulfide xHOAc 24mono-NHS ester of glutaryl-peptide 23Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH₂ cyclic (2-12) disulfide 25Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide•yTFA 26Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide•zHOAc 27Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide•X HOAc 28Mono-NHS ester of glutaryl-peptide 27 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K(NHS-Glut)]-NH₂ cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide 29DSPE-PEG2000-NH₂ Where m, n, x, y, z are variable depending onlyophilization conditions.

Referring now to FIGS. 6 and 7, in certain embodiments monomer peptidecomponents of heterodimer peptide (27), namely TFA salts compounds (22)and (25), are subjected to ion exchange chromatography on themacroporous sulfonic acid cation exchange resin AG MP-50 using a stepgradient of ammonium acetate to convert them to their acetate salts.Then the two peptide monomer acetates (23) and (26) may be tetheredthrough a glutaryl linker to form the dimer (27) as an acetate salt.Purification of the crude dimer acetate salt of (27), by C-18preparative HPLC using a linear gradient method employing CH₃CN/H₂O eachcontaining 10 mM NH₄OAc provides the pure dimer acetate (27).Conjugation of this dimer to DSPE-PEG2000-NH₂ (29) and finalpurification of the crude mixture by C-3 preparative HPLC usingCH₃CN/H₂O/NH₄OAc provides compound (21) as the acetate salt.

More specifically, compounds (22), (25) and (27) all bear side-chaincarboxylic acid and amino groups. AG MP-50, a macroporouscation-exchange resin, may be used to allow full penetration of theresin by the peptides and to exploit the immobilization of the peptidesvia their basic (amino and guanidine groups). TFA salts of the peptidesmay be adsorbed to an AG MP-50 column (sulfonic acid form) and thecolumn may be washed with water and then eluted with a step gradient ofNH₄OAc in 0 or 30% CH₃CN/H₂O, depending on the solubility of thepeptides. The peptides may be eluted at about 600 mM NH₄OAc and theacetate form of the peptides then may be obtained in pure form. Both ICfluorine analysis and CE TFA counter-ion analysis consistently show verylow TFA content of the peptides.

Preferred methods also include redissolution/relyophilization of thefinal peptides several times to remove residual NH₄OAc. Otherwise,residual traces of NH₄OAc present in the peptides may give rise to freeammonia in the presence of DIEA. This may result in the formation ofunwanted peptide-Glut-amide as a major product in subsequent preparationof (27) from the monomers (23) and (26) or final phospholipid-peptideconjugate (21) from the acetate salt of (27).

Referring now to FIG. 7, another embodiment provides the conversion ofthe TFA salt of dimer (27) to its analogous acetate salt by ion exchangechromatography on the macroporous sulfonic acid cation exchange resin AGMP-50. This dimer acetate then may be conjugated with DSPE-PEG2000-NH₂followed by purification of the crude material by C-3 preparative columnusing CH₃CN/H₂O/NH₄OAc to give the final compound (21) as an acetatesalt.

While the methods described above and in FIGS. 6 and 7 provide excellentresults, the second approach has the advantage of requiring fewer steps.Additional details are provided below in the Examples section.

Turning to FIG. 8, another embodiment provides methods for providingdimeric conjugates having minimal amounts of TFA utilizing the sizedifferential between the phospholipid-peptide conjugate (21) and TFAions. In this embodiment 21.nTFA adduct may be eluted down a sizeexclusion column in the presence of ammonium bicarbonate buffer. Thecrude 21.nTFA initially may be freed of the lyso-compound by preparativeHPLC on a Zorbax C-3 column using a linear gradient of acetonitrile intowater. Both phases may be buffered with 10 mM ammonium acetate. Thisprovides separation of the lyso-compound as indicated by analyticalHPLC.

To further reduce the amount of TFA, the material may be applied to aSephadex G-25 column and eluted with aqueous ammonium bicarbonatesolution. The eluate may be monitored by HPLC. Product-containingfractions may be pooled and lyophilized to afford the desired material(21) essentially free of TFA and with high recovery rates. Additionaldetail is provided below in the Examples section.

Both the monomeric and dimeric peptide phospholipid conjugates describedherein may be incorporated into ultrasound contrast agents such as, forexample, gas filled microvesicles. Such gas filled microvesiclesinclude, for example, gas filled microbubbles, gas filled microballoons,gas filled microcapsules, etc. In a preferred embodiment, the peptidephospholipid conjugates may be incorporated into ultrasound contrastagents comprising gas filled microbubbles. Methods of preparation of gasfilled microbubbles from phospholipids and phospholipid conjugates areknown to those skilled in the art. For example, microbubbles accordingto the present invention can be prepared by methods described in any oneof the following patents: EP 554213, WO 04/069284, U.S. Pat. No.5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No.5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833,U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No.5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat.No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S.Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No.5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S.Pat. No. 5,908,610, which are incorporated by reference herein in theirentirety. The methods disclosed in WO 04/069284 are particularlypreferred.

Suitable phospholipids include esters of glycerol with one or twomolecules of fatty acids (the same or different) and phosphoric acid,wherein the phosphoric acid residue is in turn bonded to a hydrophilicgroup, such as choline, serine, inositol, glycerol, ethanolamine, andthe like groups. Fatty acids present in the phospholipids are in generallong chain aliphatic acids, typically containing from 12 to 24 carbonatoms, preferably from 14 to 22, that may be saturated or may containone or more unsaturations. Examples of suitable fatty acids are lauricacid, myristic acid, palmitic acid, stearic acid, arachidic acid,behenic acid, oleic acid, linoleic acid, and linolenic acid. Mono estersof phospholipids are known in the art as the “lyso” forms of thephospholipid.

Further examples of phospholipids are phosphatidic acids, i.e., thediesters of glycerol-phosphoric acid with fatty acids, sphingomyelins,i.e., those phosphatidylcholine analogs where the residue of glyceroldiester with fatty acids is replaced by a ceramide chain, cardiolipins,i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid,gangliosides, cerebrosides, etc.

As used herein, the term phospholipids includes either naturallyoccurring, semisynthetic or synthetically prepared products that can beemployed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins. Examples of semisynthetic phospholipids are thepartially or fully hydrogenated derivatives of the naturally occurringlecithins.

Examples of synthetic phospholipids are e.g.,dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine(“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidyl-ethanolamin-e (“DMPE”), dipalmitoylphosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine(“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoylphosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”),distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine(“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoylsphingomyelin (“DSSP”).

Suitable phospholipids further include phospholipids modified by linkinga hydrophilic polymer thereto. Examples of modified phospholipids arephosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), inbrief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilicethanolamine moiety is linked to a PEG molecule of variable molecularweight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG,DMPE-PEG or DAPE-PEG (where DAPE is1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The compositionsalso may contain other amphiphilic compounds including, for instance,fatty acids, such as palmitic acid, stearic acid, arachidonic acid oroleic acid; sterols, such as cholesterol, or esters of sterols withfatty acids or with sugar acids; glycerol or glycerol esters includingglycerol tripalmitate, glycerol distearate, glycerol tristearate,glycerol dimyristate, glycerol trimyristate, glycerol dilaurate,glycerol trilaurate, glycerol dipalmitate; tertiary or quaternaryalkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane(DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), andmixtures or combinations thereof.

Preferably, the formulation comprises at least one component bearing anoverall net charge, such as, for instance, phosphatidic acid, PE-PEG,palmitic acid, stearic acid, Ethyl-DSPC or DSTAP, preferably in a molaramount of less than about 50%. Particularly preferred formulations mayinclude mixtures of two or more of the following components: DSPC, DPPG,DPPA, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid. Somepreferred phospholipids and formulations are set forth in the examplesAny of the gases disclosed herein or known to the skilled artisan may beemployed; however, inert gases, such as SF₆ or perfluorocarbons likeCF₄, C₃F₈ and C₄F₁₀, are preferred, optionally in admixture with othergases such as air, nitrogen, oxygen or carbon dioxide

The preferred microbubble suspensions of the present invention may beprepared from phospholipids using known processes such as afreeze-drying or spray-drying solutions of the crude phospholipids in asuitable solvent or using the processes set forth in EP 554213; WO04/069284; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962;EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat.No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No.5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat.No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S.Pat. No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No.5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat.No. 6,187,288; and U.S. Pat. No. 5,908,610, which are incorporated byreference herein in their entirety. Preferably, as disclosed inInternational patent application WO 04/069284, a microemulsion can beprepared which contains the phospholipids (e.g DSPC and/or DSPA) inadmixture with a lyoprotecting agent (such as, for instance,carbohydrates, sugar alcohols, polyglycols and mixtures thereof, asindicated in detail hereinafter) and optionally other amphiphilicmaterials (such as stearic acid), dispersed in an emulsion of water andof a water immiscible organic solvent. Preferred organic solvents arethose having solubility in water of 1.0 g/l or lower, preferably lowerabout 0.01 g/l, and include, for instance, pentane, hexane, heptane,octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane,cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene,ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl etherand di-isopropylketone, chloroform, carbon tetrachloride,2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether,ethylperfluorobutylether, ethylperfluoroisobutylether and mixturesthereof. The peptide-phospholipid conjugate of the invention can beadmixed together with the phospholipid forming the microvesicle'senvelope, in the microemulsion. Preferably, an aqueous suspension of thepeptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000) isfirst prepared, which is then admixed together with an aqueous-organicemulsion comprising the phospholipid and the lyoprotecting agent.Preferably said mixing is effected under heating, e.g. form about 40° C.to 80° C.

Prior to formation of the suspension of microbubbles by dispersion in anaqueous carrier, the freeze dried or spray dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles that are stable even whenstored for prolonged periods and are obtained by simple dissolution ofthe dried laminarized phospholipids (which have been stored under adesired gas) without shaking or any violent agitation.

Alternatively, microbubbles can be prepared by suspending a gas into anaqueous solution at high agitation speed, as disclosed e.g. in WO97/29783. A further process for preparing microbubbles is disclosed inWO 2004/069284, herein incorporated by reference, which comprisespreparing an emulsion of an organic solvent in an aqueous medium in thepresence of a phospholipid and subsequently lyophilizing said emulsion,after optional washing and/or filtration steps. Some preferredpreparation methods are disclosed in the examples.

The formulation for the preparation of the gas-filled microbubbles mayadvantageously further comprise a lyophilization additive, such as anagent with cryoprotective and/or lyoprotective effect and/or a bulkingagent, for example an amino-acid such as glycine; a carbohydrate, e.g. asugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose ora cyclodextrin, or a polysaccharide such as dextran; or a polyglycolsuch as polyethylene glycol (e.g. PEG-4000).

Any of these ultrasound compositions should also be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents may be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions may include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas may be used in the ultrasound contrast agents ofthe invention. The term “gas” as used herein includes any substances(including mixtures) substantially in gaseous form at the normal humanbody temperature. The gas may thus include, for example, air, nitrogen,oxygen, CO₂, argon, xenon or krypton, fluorinated gases (including forexample, perfluorocarbons, SF₆, SeF₆) a low molecular weight hydrocarbon(e.g., containing from 1 to 7 carbon atoms), for example, an alkane suchas methane, ethane, a propane, a butane or a pentane, a cycloalkane suchas cyclopropane, cyclobutane or cyclopentene, an alkene such asethylene, propene, propadiene or a butene, or an alkyne such asacetylene or propyne and/or mixtures thereof. However, fluorinated gasesare preferred. Fluorinated gases include materials that contain at leastone fluorine atom such as SF₆, freons (organic compounds containing oneor more carbon atoms and fluorine, i.e., CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀,CBrF₃, CCl₂F₂, C₂ClF₅, and CBrClF₂) and perfluorocarbons. The termperfluorocarbon refers to compounds containing only carbon and fluorineatoms and includes, in particular, saturated, unsaturated, and cyclicperfluorocarbons. The saturated perfluorocarbons, which are usuallypreferred, have the formula C_(n)F_(n)+2, where n is from 1 to 12,preferably from 2 to 10, most preferably from 3 to 8 and even morepreferably from 3 to 6. Suitable perfluorocarbons include, for example,CF₄, C₂F₆, C₃F₈C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Mostpreferably the gas or gas mixture comprises SF₆ or a perfluorocarbonselected from the group consisting of C₃F₈C₄F₈, C₄F₁₀, C₅F₂, C₆F₁₂,C₇F₁₄, C₈F₁₈, with C₄F₁₀ being particularly preferred. See also WO97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO98/18497, WO 98/18501, WO 98/05364, WO 98/17324. In a preferredembodiment the gas comprises C₄F₁₀ or SF₆, optionally in admixture withair, nitrogen, oxygen or carbon dioxide.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferablythe gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor may be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons may beused as temperature activated gas precursors. These perfluorocarbons,such as perfluoropentane, have a liquid/gas phase transition temperatureabove room temperature (or the temperature at which the agents areproduced and/or stored) but below body temperature; thus they undergo aphase shift and are converted to a gas within the human body.

As discussed above, the gas can comprise a mixture of gases. Thefollowing combinations are particularly preferred gas mixtures: amixture of gases (A) and (B) in which, at least one of the gases (B),present in an amount of between 0.5-41% by vol., has a molecular weightgreater than 80 daltons and is a fluorinated gas and (A) is selectedfrom the group consisting of air, oxygen, nitrogen, carbon dioxide andmixtures thereof, the balance of the mixture being gas A.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized product may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably insuch a case it can be supplied in the form of a two-component kit, whichcan include two separate containers or a dual-chamber container. In theformer case preferably the container is a conventional septum-sealedvial, wherein the vial containing the lyophilized residue of step b) issealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent. In both casesmeans for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided. However,as noted above, in the stabilised contrast agents according to theinvention the size of the gas microbubbles is substantially independentof the amount of agitation energy applied to the reconstituted driedproduct. Accordingly, no more than gentle hand shaking is generallyrequired to give reproducible products with consistent microbubble size.

It can be appreciated by one of ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific containers, vial or connection systems are required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirable substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.The stopper may be a compound or multicomponent formulation based on anelastomer, such as poly(isobutylene) or butyl rubber.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles can be administered, for example, in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.

Ultrasound imaging techniques that can be used in accordance with thepresent invention include known techniques, such as color Doppler, powerDoppler, Doppler amplitude, stimulated acoustic imaging, and two- orthree-dimensional imaging techniques. Imaging may be done in harmonic(resonant frequency) or fundamental modes, with the second harmonicpreferred.

The ultrasound contrast agents of the present invention may further beused in a variety of therapeutic imaging methods. The term therapeuticimaging includes within its meaning any method for the treatment of adisease in a patient which comprises the use of a contrast imaging agent(e.g. for the delivery of a therapeutic agent to a selected receptor ortissue), and which is capable of exerting or is responsible to exert abiological effect in vitro and/or in vivo. Therapeutic imaging mayadvantageously be associated with the controlled localized destructionof the gas-filled microvesicles, e.g. by means of an ultrasound burst athigh acoustic pressure (typically higher than the one generally employedin non-destructive diagnostic imaging methods). This controlleddestruction may be used, for instance, for the treatment of blood clots(a technique also known as sonothrombolysis), optionally in combinationwith the localized release of a suitable therapeutic agent.Alternatively, said therapeutic imaging may include the delivery of atherapeutic agent into cells, as a result of a transient membranepermeabilization at the cellular level induced by the localized burst ofthe microvesicles. This technique can be used, for instance, for aneffective delivery of genetic material into the cells; optionally, adrug can be locally delivered in combination with genetic material, thusallowing a combined pharmaceutical/genetic therapy of the patient (e.g.in case of tumor treatment).

The term “therapeutic agent” includes within its meaning any substance,composition or particle which may be used in any therapeuticapplication, such as in methods for the treatment of a disease in apatient, as well as any substance which is capable of exerting orresponsible to exert a biological effect in vitro and/or in vivo.Therapeutic agents thus include any compound or material capable ofbeing used in the treatment (including diagnosis, prevention,alleviation, pain relief or cure) of any pathological status in apatient (including malady, affliction, disease lesion or injury).Examples of therapeutic agents are drugs, pharmaceuticals, bioactiveagents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents,proteins, natural or synthetic peptides, including oligopeptides andpolypeptides, vitamins, steroids and genetic material, includingnucleosides, nucleotides, oligonucleotides, polynucleotides andplasmids.

Materials and Analytical Methods

Solvents for reactions, chromatographic purification and HPLC analyseswere E. Merck Omni grade solvents from VWR Corporation (West Chester,Pa.). N-Methylpyrrolidinone (NMP) and N,N-dimethylformamide (DMF) wereobtained from Pharmco Products Inc. (Brookfield, Conn.), and werepeptide synthesis grade or low water/amine-free Biotech grade quality.Piperidine (sequencing grade, redistilled 99+%) and trifluoroacetic acid(TFA) (spectrophotometric grade or sequencing grade) were obtained fromSigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka ChemicalDivision of Sigma-Alrich Corporation. N,N′-Diisopropylcarbodiimide(DIC), phenol (99%), N,N-diisopropylethylamine (DIEA) andtriisopropylsilane (TIS) were purchased from Sigma-Aldrich Corporation.Fmoc-protected amino acids, pseudoproline dipeptides,Fmoc-Asp(O-tBu)-Ser(ψ^(Me,Me)pro)-OH and Fmoc-Gly-Thr(ψ^(Me,Me)pro)-OHand N-hydroxybenzotriazole (HOBt) were obtained from Novabiochem (SanDiego, Calif.). Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtainedfrom NeoMPS Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad,India). Disuccinimidyl glutarate (DSG) and1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2000-NH₂] wereobtained from Pierce Chemical Co. (Rockford, Ill.) and Avanti® PolarLipids (Alabaster, Ala.), respectively. Fmoc-Gly-Gly-Gly-OH andFmoc-Gly-Gly-OH were prepared in-house from the corresponding triglycineor diglycine by the reaction with Fmoc-OSu. An AG MP-50 ion-exchangeresin was obtained from Bio-Rad (Hercules, Calif.).

Analytical HPLC data were generally obtained using a Shimadzu LC-10AT VPdual pump gradient system employing a Waters XTerra MS-C18 4.6×50 mmcolumn, (particle size: 5μ; 120 Å pore size) and gradient or isocraticelution systems using water (0.1% TFA) as eluent A and CH₃CN (0.1% TFA)or CH₃CN—CH₃OH (1:1, v/v) (0.1% TFA) as eluent B. Detection of compoundswas accomplished using UV at 220 and 254 nm. The purity of thephospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5 μM,300 Å, 4.6×250 mm) column or on a Zorbax 300 SB-C3 (3.5 μM; 300 Å, 3×150mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with aUV detector.

Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradientsystem equipped with a SPD-10AV UV detector fitted with a preparativeflow cell. Generally the solution containing the crude peptide wasloaded onto a reversed phase C18, C4 or C3 column, depending on thecompound characteristics, using a third pump attached to the preparativeShimadzu LC-8A dual pump gradient system. After the solution of thecrude product mixture was applied to the preparative HPLC column thereaction solvents and solvents employed as diluents, such as DMF orDMSO, were eluted from the column at low organic phase composition. Thenthe desired product was eluted using a gradient elution of eluent B intoeluent A. Product-containing fractions were combined based on theirpurity as determined by analytical HPLC and mass spectral analysis. Thecombined fractions were freeze-dried to provide the desired product.

Amino acid composition analyses were performed at the Keck BiotechnologyResource Laboratory at Yale University, New Haven, Conn. Mass spectraldata were obtained from MScan Inc. (606 Brandywine Parkway, West ChesterPa. 19380) or obtained in-house on an Agilent LC-MSD 1100 MassSpectrometer. For the purposes of fraction selection andcharacterization of the products mass spectral values were usuallyobtained using API-ES in negative ion mode. Generally the molecularweight of the target peptides was ˜3000; the mass spectra usuallyexhibited doubly or triply negatively charged ion mass values ratherthan [M-H]⁻. These were generally employed for selection of fractionsfor collection and combination to obtain the pure peptide during HPLCpurification. In some cases fractions exhibited dominant peaksattributable to [M-2H]/2+57 or [M-2H]/2+114 in the mass spectrum. Thesepeaks are due to the formation of adducts of one or two molecules oftrifluoroacetic acid per molecule of the peptide. After carefulcollection of fractions by comparing MS results and HPLC purities andfreeze-drying process, a small amount of the isolated fluffy solid wasdissolved in water (0.5 mg/mL) and treated with a drop of aqueousN-methyl-D-glucamine (˜0.5 M). This solution was analyzed by HPLC and MSfor final purity results of the purified peptide. Peptide solutions inthe presence of N-methyl-D-glucamine did not exhibit [M-2H]/2+57 or[M-2H]/2+114 mass value peaks in the mass spectrum, instead the expected[M-2H]/2 or [M-3H]/3 peaks were observed.

The following non-limiting Examples provide additional detail onefficient processes used for obtaining large quantities of highlypurified forms of the monomeric and dimeric peptide phospholipidconjugates. These non-limiting Examples also describe the preparation ofrepresentative targeted microbubbles which include these monomeric anddimeric peptide phospholipid conjugates. These Examples also describethe use of such targeted microbubbles in static binding tests onKDR-transfected cells and dynamic binding tests on rh VEGF-R2/Fcchimeric protein. The Examples further describe the evaluation ofultrasound contrast agents containing KDR binding lipopeptides in arabbit VX2 tumor model.

EXAMPLES

Examples 1-2 below refer to the monomeric peptide phospholipid conjugateshown in FIG. 2. A process for synthesizing this compound is shown inFIG. 1. Although these Examples refer more specifically to the processfor synthesizing the compound shown in FIG. 2, similar processes mayused to prepare the monomeric peptide phospholipid conjugate shown inFIG. 10 and the linear peptide monomer (32) shown in FIG. 9 as well asother monomer peptide-phospholipid conjugates including, for example,those formed using peptide monomers Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2(SEQ. ID NO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH₂ (SEQ. ID NO.7), as well as analogs and derivatives of any of the above.Additionally, co-pending U.S. application Ser. No. 10/661,156, filedSep. 11, 2003, sets forth methods for the preparation of the peptidemonomers and is incorporated by reference herein in its entirety.

Example 1

Solid Phase Synthesis (SPPS) and Purification ofLinear Peptide Monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂, (SEQ IDNO. 2) Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH₂,; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide

The linear peptide monomer (2) was synthesized by an establishedautomated protocol on a SONATA®/Pilot Peptide Synthesizer usingFmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids andDIC-mediated HOBt ester activation in DMF. The peptide sequence wassynthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PSresin, typically on a 10 mmol scale. The amino acid couplings werecarried out with a 4-fold excess each of amino acid and the DIC-HOBtreagent pair in DMF.

In a typical coupling of an amino acid, 5 mL of dry DMF per gram ofresin was used. The total volume of DMF, calculated on the basis ofresin used, was allocated among amino acid, HOBt and DIC for solutionpreparation. For example, for the synthesis involving 50 g (10 mmolscale) of resin, the calculated volume of 250 mL of DMF was distributedamong amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acidvessel on the Sonata Pilot Peptide Synthesizer was charged with thesolid dry amino acid (4-fold excess with respect to the resin). Atinception of the coupling step, the software of the instrument wasemployed to deliver successively the chosen volume of DMF (for dilutionof the amino acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF andmixing by nitrogen bubbling was initiated and conducted for 4 min. Thisserved to pre-activate the amino acid and to insure complete dissolutionof all components of the mixture. After activation, the softwaremediated the transfer of the solution of the activated Fmoc-amino acidto the reaction vessel containing the resin. After transfer was completethe vessel was agitated for 3 h with recurrent nitrogen bubbling. Afterthe 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g,6×) and the cleavage of the Fmoc-group was performed with 25% piperidinein DMF (5 mL/g) containing HOBt (0.1M) (2×10 min). The resin wasthoroughly washed with DMF (5 mL/g, 6×) to assure complete removal ofpiperidine from the resin in preparation for the ensuing amino acidcoupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, thepre-activation in the amino acid bottle was not conducted in order tominimize the formation of diketopiperazine during the activation time asdiscussed in the text. Therefore, in these two cases, the solutions ofamino acid, HOBt and DIC were added to the reaction vessel sequentiallyand the coupling process was conducted with ‘in situ’ activation.

After chain elongation was completed, the Fmoc group of the N-terminalamino acid was removed in the standard manner followed by the standardwash with DMF (vide supra). The N-terminal amino acid was then capped bytreatment with freshly prepared acetylation mixture (0.5M aceticanhydride, 0.125M DIEA and 0.015M HOBt in DMF/6 mL/g of resin), 2×20min. After completion of the peptide synthesis, the resin was treatedwith the cleavage cocktail, ‘Reagent B’(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g ofresin) for 4 h. The volatiles were removed and the paste thus obtainedwas triturated with ether to provide a solid which was washed with ether(3×) with intervening centrifugation (to compact the suspended solids inorder to allow decantation of the supernatant) and then dried undervacuum to provide the required peptide as an off-white solid. A 10 mmolscale synthesis of the linear peptide monomer (2) gave 33.82 g (103% oftheory) of the crude peptide. The greater than theoretical yield wasmost likely due to moisture and residual solvents.

Purification of the Linear Peptide Monomer (2)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH₂ (SEQ ID NO.2); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH₂; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide

A ˜0.5 g portion of the crude linear peptide monomer (2) was dissolvedin a minimum amount of CH₃CN (˜20 mL). The volume of the solution wasadjusted to 100 mL with water and employing a third pump the solutionwas loaded onto a reversed phase C18 preparative column (Waters, XTerra®Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) which had beenpre-equilibrated with 10% CH₃CN in water (0.1% TFA). The column was noteluted with the equilibrating eluent during application of the samplesolution. After the sample solution was applied to the column, thecomposition of the eluent was ramped to 20% CH₃CN-water (0.1% TFA) over1 min, and a linear gradient at a rate of 0.6%/min of CH₃CN (0.1% TFA)into water (0.1% TFA) was initiated and maintained for 50 min. Fractions(15 mL) were manually collected using UV at 220 nm as an indicator ofproduct elution. The collected fractions were analyzed on a WatersXTerra analytical reversed phase C-18 column (5μ particle, 120 Å pore)and product-containing fractions of >95% purity were pooled andfreeze-dried to afford the corresponding pure linear peptide monomer(2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g(24% yield) of the desired product (>95% purity).

Example 2

Preparation of Monomeric Peptide Phospholipid Conjugate (1)Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ ID NO. 1); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH₂; N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-l-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide

The monomeric peptide phospholipid conjugate (1),Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH₂ (SEQ ID NO.1), was prepared by conjugation of (3), the glutaric acid monoamidemono-NHS ester of peptide monomer (2), with DSPE-PEG2000-NH₂phospholipid ammonium salt (4).

A round-bottomed flask equipped with magnetic stir bar and septum capwas charged sequentially with anhydrous dimethylformamide (7.5 mL),disuccinimidyl glutarate (DSG, 0.25 g, 0.75 mmol) anddiisopropylethylamine (0.10 g, 0.78 mmol) with stirring. Solid linearpeptide monomer (2) (0.5 g, 0.152 mmol) was added portionwise to theabove solution over a period of 2 min; then the solution was stirred for30 min at ambient temperature. The reaction mixture was diluted to ˜50mL with anhydrous ethyl acetate; this resulted in precipitation of theintermediate mono-NHS ester (3), the glutaric acid monoamide mono-NHSester of peptide monomer (2). The solution was centrifuged to bring downmono-NHS ester (3)) as a colorless solid. The supernatant containingexcess DSG was decanted from the compacted solid mono-NHS ester (3)which was again dispersed in ethyl acetate, centrifuged and washed twicemore to remove the remaining traces of DSG. The solid intermediatemono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and themixture was stirred.

Meanwhile, DSPE-PEG2000-NH₂ phospholipid ammonium salt (4) (0.38 g, 0.14mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separateflask and trifluoroacetic acid (2 drops) was added to protonate thephosphodiester oxygen facilitating solubilization of phospholipidammonium salt in dichloromethane. The clear solution was then evaporatedon a rotary evaporator to remove the volatiles and dried further undervacuum.

The solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) andtransferred to the stirred solution of mono-NHS ester (3) and theresulting mixture was stirred for 24 h at ambient temperature. Thereaction mixture was diluted to 100 mL with a 1:1 mixture of CH₃OH andCH₃CN-water (1:1, v/v) and the insolubles were filtered. Half of thefiltered solution was loaded onto a reversed phase C2 preparative column(Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm) which had beenpre-equilibrated with 3:1 (v/v) mixture of water (0.1% TFA) andCH₃OH—CH₃CN (1:1, v/v, 0.1% TFA) at a flow rate of 100 mL/min. Note thatthe column was not eluted with the equilibrating eluent during loadingof the sample. After the sample solution was loaded the column waswashed with the equilibration eluent until the plug of DMF was eluted.The composition of the eluent was ramped to 70% CH₃OH—CH₃CN (1:1, 0.1%TFA) over 9 min and a linear gradient of 0.75%/min of CH₃OH—CH₃CN (1:1,0.1% TFA) into water (0.1% TFA) was initiated and run for 40 min.Fractions (15 mL) were collected using UV (220 nm) as an indicator ofproduct elution. Fractions were checked for purity on an analytical HPLCsystem (column: YMC C-4, 5μ, 300 Å, 4.6×250 mm) using UV at 220 nm andan evaporative light scattering detector (ELSD). The latter detector(ELSD) was employed to detect DSPE-PEG2000-NH₂ phospholipid ammoniumsalt (4) which has very little UV absorbance at 220 nm.Product-containing fractions of >98% purity, and devoid ofDSPE-PEG2000-NH₂ phospholipid ammonium salt (4) were combined andconcentrated on a rotary evaporator to reduce the content of CH₃OH. Theconcentrated solution was then diluted with 10% CH₃CN in water until afaint flocculent precipitate formed. The resulting solution wasfreeze-dried to provide monomeric peptide phospholipid conjugate (1) asa colorless solid. The second portion of crude monomeric peptidephospholipid conjugate (1) was purified as described above. The combinedyield of the target monomeric peptide phospholipid conjugate (1) was0.40 g (47% yield).

Examples 3-5 below refer to the dimeric peptide phospholipid conjugateshown in FIG. 5. Representative methods of synthesizing the dimericconjugate are shown in FIGS. 3, 4, 6, 7 and 8.

Example 3

Solid Phase Synthesis (SPPS), Cyclization andPurification of Monomer Peptides (12)Ac-AGPTWC*LDDWYYC*WLFGTGGGK[K(ivDde)]-NH₂ and (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂

The linear peptides were synthesized by an established automatedprotocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PSresin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBtester activation in DMF. The peptide sequence on the Fmoc-Pal-Peg-PSresin was synthesized in stepwise fashion by SPPS methods typically on a10 mmol scale. The amino acid coupling was carried out with a 4-foldexcess each of amino acid and DIC-HOBt reagent in DMF.

In a typical coupling of an amino acid in the sequence, 5 mL of dry DMFper gram of resin was used. The total volume of DMF, calculated on thebasis of resin used, was allocated among amino acid, HOBt and DIC forsolution preparation. For example, for the synthesis involving 50 g ofresin, the calculated volume of 250 mL of DMF was distributed amongamino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vesselon the Sonata® Pilot Peptide Synthesizer was charged with the solid dryamino acid (4-fold excess with respect to the resin). At inception ofthe coupling step, the chosen volume of DMF and HOBt (4 eq.) in DMF andDIC (4 eq.) in DMF were delivered successively and after each deliverymixing by nitrogen bubbling was conducted. After the last reagent wasdelivered mixing by nitrogen bubbling was initiated and conducted for 4min. This served to preactivate the amino acid and to insure completedissolution of all components of the mixture.

After activation, the solution of the activated Fmoc-amino acid wastransferred to the reaction vessel containing the resin. After transferwas complete the vessel was agitated for 3 h with recurrent nitrogenbubbling. After the 3 h coupling time, the resin was washed thoroughlywith DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performedwith 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2×10 min).The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure completeremoval of piperidine from the resin in preparation for the ensuingamino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH andFmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was notconducted in order to minimize the formation of diketopiperazine duringthe activation time as discussed in the text. Therefore, in these twocases, the solution of the amino acid, HOBt and DIC were added to thereaction vessel sequentially and the coupling process was conducted with‘in situ’ activation. After chain elongation was completed, the fmocgroup of the N-terminal amino acid was removed in the standard mannerfollowed by the standard wash with DMF (vide supra). The N-terminalamino acid was then capped by treatment with freshly preparedacetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBtin DMF-6 mL/g of resin), 2×20 min.

Functionalization of the ε-amino group of C-terminal Lysine moieties ofthe monomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) asrequired) was accomplished by first removing the ivDde group of theε-amino group with freshly prepared 10% hydrazine in DMF (5 mL/g ofresin—2×10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) thecoupling time was increased to 10 h. After completion of the peptidesynthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’(TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g ofresin) for 4 h. After evaporation of the volatiles under vacuum, thepaste was triturated with ether to provide a solid which was collectedby filtration washed with diethyl ether and dried. A 10 mmol scalesynthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH₂ gave 30 g(103% of theory) of the crude peptide. In the case of (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂, a 10 mmol scale synthesisgave 28 g (107% of theory) of crude peptide. The greater thantheoretical yields are most likely due to moisture and residualsolvents.

Cyclization of the Linear Di-Cysteine Peptides to Cyclic DisulfidePeptides

Cyclic disulfide peptides were prepared from the corresponding lineardi-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5,v/v). The crude linear peptide was dissolved in the solvent mixture (5mL/g) in a wide mouth beaker, and the pH of the solution was adjusted to8.5 by the addition of solid N-methyl-D-glucamine in portions. Theresulting mixture was stirred for 36 h at ambient temperature. Thesolution was then diluted with acetonitrile (50 mL/g) and the mixturewas stirred for 2 min. The solid cyclic disulfide peptide was collectedby filtration, washed with diethyl ether and dried.

Purification of Monomer Peptides

Peptide Monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH₂; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH₂ cyclic (6-13) disulfide

A ˜0.5 g portion of the crude cyclic disulfide peptide monomer (12) wasdissolved in a minimum amount of DMSO (˜3 mL). The volume of thesolution was adjusted to ˜100 mL with 20% CH₃CN-water and employing athird pump, the solution was loaded onto a reversed phase C18preparative column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm,flow rate 100 mL/min), which had been pre-equilibrated with 10% CH₃CN inwater (0.1% TFA). During application of the sample solution to thecolumn the flow of the equilibrating eluent from the preparative HPLCsystem was stopped. After the sample solution was applied to the column,the flow of equilibrating eluent from the gradient HPLC system wasreinitiated and the column was eluted with 10% CH₃CN-water (0.1% TFA)until the DMSO was eluted. Then the eluent composition was ramped to 35%CH₃CN-water (0.1% TFA) over 1 min after which a linear gradient at arate of 0.5%/min CH₃CN (0.1% TFA) into water (0.1% TFA) was initiatedand maintained for 50 min. Fractions (15 mL) were manually collectedusing UV at 220 nm as an indicator of product elution. The collectedfractions were analyzed on a Waters XTerra analytical reversed phaseC-18 column (5μ particle, 120 Å pore) and product-containing fractionsof >95% purity were pooled and freeze-dried to afford the correspondingcyclic disulfide peptide monomer (12). Typically the purification of 0.5g of crude peptide monomer (12) afforded 0.1 g (20% yield) of thedesired product (>95% purity).

Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂; Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH₂ cyclic (2-12) disulfide

Following the procedure employed for the HPLC purification of peptidemonomer (2), the crude cyclic disulfide peptide monomer (13)Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂ (0.5 g) dissolved in 20%CH₃CN-water mixture (100 mL) was loaded onto a reversed phase C18preparative column (Waters, XTerra® Prep MS C18, 50×250 mm, 101particle, 300 Å pore, flow rate 100 mL/min), which had beenpre-equilibrated with 10% CH₃CN (0.1% TFA) in water (0.1% TFA). Duringapplication of the sample solution to the column the flow of theequilibrating eluent from the preparative HPLC system was stopped. Afterthe sample solution was applied to the column, the flow of equilibratingeluent from the gradient HPLC system was reinitiated and the column waseluted with 10% CH₃CN-water (0.1% TFA) for 5 min. Then the eluentcomposition was ramped to 30% CH₃CN (0.1% TFA)-water (0.1% TFA) over 1min and a linear gradient elution at a rate of 0.5%/min of CH₃CN (0.1%TFA) into water (0.1% TFA) was initiated and maintained for 50 min.Fractions (15 mL) were manually collected using UV at 220 nm as anindicator of product elution. The fractions were analyzed on a WatersXTerra analytical reversed phase C-18 column (4.6 mm i.d.×50 mm, 5μparticle, 120 Åpore) and product-containing fractions of >95% puritywere pooled and freeze-dried to afford the corresponding cyclicdisulfide peptide monomer (13). Typically the purification of 0.5 g ofcrude peptide monomer (3) afforded 0.12 g (24% yield) of the desiredproduct (>95% purity).

Example 4

Preparation and Purification of Precursor Dimer Peptide (16)Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH₂ cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-Lys)]-NH₂ cyclic(2-12) disulfide ]-NH₂ cyclic (6-13) disulfide

As shown in FIG. 3, disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol)was dissolved in stirred anhydrous dimethylformamide (2.0 mL) anddiisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Thensolid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH₂(0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSGover a period of two min. After stirring for 30 min at room temperature,the solution was diluted with anhydrous ethyl acetate to 50 mL, (thisserved to precipitate intermediate mono-NHS ester (14)). The entiremixture was centrifuged and the supernatant was decanted leavingintermediate mono-NHS ester (14) as a colorless solid. The solid wasresuspended with ethyl acetate; the solution containing the suspendedsolid mono-NHS ester (14) was centrifuged to separate the solid and thesupernatant was again decanted. This washing process was repeated twiceto remove completely the excess DSG.

The solid mono-NHS ester (14) was dissolved in stirred anhydrousdimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol)was added. Then solid peptide monomer (13),Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH₂, (0.50 g, 0.19 mmol, 1.12 eq.)was added in portions to the stirred solution over a three min. periodand the resulting mixture was stirred for 18 h. The reaction wasmonitored by mass spectrometry; after the complete consumption of thepeptide monomer glutaric acid monoamide mono-NHS ester (14) wasconfirmed, neat hydrazine (0.1 mL) was added to remove the ivDdeprotecting group of the ivDde-bearing dimer (15) and the mixture wasstirred for 20 min at room temperature.

The solution was then acidified by dropwise addition of TFA and themixture was diluted to 100 mL with 10% CH₃CN (0.1% TFA) in water (0.1%TFA). The solution was filtered to remove particulates and half of theclarified solution was loaded onto a reversed phase C18 preparativecolumn (Waters, XTerra® Prep MS C18, 10μ, 50×250 mm, flow rate 100mL/min) pre-equilibrated with 10% CH₃CN in water (0.1% TFA). Duringapplication of the sample solution to the column the flow of theequilibrating eluent from the preparative HPLC system was stopped. Afterthe sample solution was applied to the column, the flow of equilibratingeluent from the gradient HPLC system was reinitiated and the column waseluted with 10% CH₃CN-water (0.1% TFA) in order to flush DMF from thecolumn. After elution of the DMF plug was completed the eluentcomposition was increased to 20% CH₃CN over one min. and the elution wascontinued with a linear gradient rate of 0.6%/min of CH₃CN (0.1% TFA)into water (0.1% TFA). Fractions (15 mL) were collected using UV (220nm) as an indicator of product elution. The fractions were analyzed on areversed phased C18 column (Waters MS C18, 4.6 mm i.d.×50 mm, 5μparticle, 120 Å pore) and the product-containing fractions of >95%purity were pooled and freeze-dried to provide precursor dimer peptide(16) as a colorless, fluffy solid. The remaining crude precursor dimerpeptide (16) was purified in the same manner. From 0.5 g each of monomerpeptides (12) and (13), 320 mg (overall yield 33%) of the desired dimer(16) was obtained (>95% purity).

Example 5

Preparation of KDR-Binding Dimeric Peptide Phospholipid Conjugate (11)Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-l-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide;AC-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDP-GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH₂cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide;Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys{Ac-Val-Cys-Tip-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH-Glut)-]-NH₂cyclic (2-12) disulfide}-NH₂ cyclic (6-13) disulfide.

The KDR-binding dimer (11) may be prepared by conjugation of precursordimer peptide (16),Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[-NH₂cyclic (2-12) disulfide]-NH₂ cyclic (6-13) disulfide, withDSPE-PEG2000-NH₂ phospholipid ammonium salt (18) as shown in FIG. 4.

Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was addedportionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF(3.0 mL) with stirring over a period of 3 min. Then the solution wasstirred at ambient temperature for 30 min. The reaction mixture wasdiluted to 50 mL with anhydrous ethyl acetate; this resulted inprecipitation of the dimer glutaric acid monoamide mono-NHS ester (17),the glutaric acid monoamide mono-NHS ester of the precursor dimerpeptide (16). The solution was centrifuged to pellet 6 (m/z, neg. ion,1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid.The supernatant ethyl acetate layer containing excess DSG was decantedfrom the compacted solid dimer glutaric acid monoamide mono-NHS ester(17) which was again resuspended in ethyl acetate, centrifuged andwashed twice more to remove the remaining traces of DSG. The solidintermediate glutaric acid monoamide mono-NHS ester dimer derivative(17) thus obtained was dissolved in anhydrous DMF/CH₂Cl₂ (8:2, v/v) (3.0mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and thesolution was stirred.

Meanwhile, DSPE-PEG2000-NH₂ phospholipid ammonium salt (18) (0.235 g,0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in aseparate flask and TFA (2 drops) was added to protonate thephosphodiester oxygen, facilitating solubilization of phospholipidammonium salt (18) in dichloromethane. The clear solution wasconcentrated to remove the volatiles and dried further under vacuum.

The solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL)and transferred to the stirred solution of glutaric acid monoamidemono-NHS ester dimer derivative (17) and the resulting mixture wasstirred for 24 h at ambient temperature. The reaction mixture wasdiluted with a solution of 50% CH₃OH, 25% CH₃CN and 25% water (1:1) to100 mL and the insolubles were filtered. Half of the filtered solutionwas loaded onto a reverse phased C4 preparative column (Kromasil® PrepC4, 10μ, 300 Å, 50×250 mm) which had been pre-equilibrated with 1:1mixture of CH₃OH and CH₃CN (1:1, 0.1% TFA) and water (0.1% TFA) at aflow rate of 100 mL/min. During application of the sample solution tothe column the flow of the equilibrating eluent from the preparativeHPLC system was stopped. After the sample solution was loaded the flowof the equilibrating eluent was reinitiated and the column was washeduntil the plug of DMF was eluted.

The composition of the eluent was then ramped to 70% CH₃OH—CH₃CN (1:1,0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient of 0.75%/minof CH₃OH—CH₃CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated. Theelution was continued after reaching 100% B in order to achieve completeelution of the product from the column. Fractions (15 mL) were collectedusing UV (220 nm) as an indicator of product elution and after the mainproduct was eluted fraction collection was continued for several minutesin order to insure elution of trace amounts of starting phospholipidammonium salt (18). Fractions were checked for purity on an analyticalHPLC system (column: YMC C4, 5 μM, 300 Å, 4.6×250 mm) using UV at 220 nmand an evaporative light scattering detector (ELSD). The latter detectoris employed to detect DSPE-PEG2000-NH₂ which has a weak chromophore at220 nm. Product-containing fractions of >98% purity, and devoid ofDSPE-PEG2000-NH₂ phospholipid ammonium salt (8) were combined andconcentrated to reduce the content of CH₃OH. The solution was thendiluted with 10% CH₃CN in water until a faint flocculent precipitateformed. The resulting solution was freeze-dried to afford the dimericpeptide phospholipid conjugate (11) as a colorless solid. The secondportion of crude dimeric peptide phospholipid conjugate (11) waspurified as described above. The combined yield of the target dimericpeptide phospholipid conjugate (11) was 0.39 g (57% yield). The samplesof the dimeric peptide phospholipid conjugate (11) made from differentsample purification runs were pooled together, dissolved intert-butanol-acetonitrile-water mixture and re-lyophilized to providethe dimeric peptide phospholipid conjugate (11) as a colorless, fluffysolid which was further dried under vacuum.

Examples 6-8 below refer to the preparation of the dimerpeptide-phospholipid conjugate shown in FIG. 5, wherein the dimericconjugate contains very low levels of TFA. FIGS. 6-8 illustrate themethods described in the Examples below.

Example 6 Preparation of Dimeric Conjugate having Low TFA Levels Via theUse of a Glutaryl Linker

Preparation of (23), (26) and Dimer Peptide (27) Acetate Salt byConversion of (22), (25) and Dimer Peptide 27.nTFA Salts to Acetates byAG MP-50 Ion-Exchange Resin

For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL resin bed)was suspended in 20% of CH₃CN/H₂O. The suspension was packed in a 3×30cm glass column and the final volume was 150 mL. The column wasconnected to a pump and a conductivity meter. It was washed with 20% ofCH₃CN/H₂O at 17 mL/min flow rate until the conductivity was below 1μs/cm. Compound (22) (210 mg) was dissolved in 20% of CH₃CN/H₂O (80 mL)and the resulting solution was loaded to the column. The column waswashed again with the same eluent until its conductivity was below 1μs/cm. A gradient of NH₄OAc in 20% of CH₃CN/H₂O was applied at 200 mM,400 mM, 600 mM and 800 mM, 250 mL each. The compound came out at 600 mMNH₄OAc. The fractions were analyzed by HPLC and the ones containing thecompound were combined and lyophilized several times until the weight ofthe material was constant. 176 mg of the pure material (23) was obtainedas a white fluffy solid. The yield was 83.8%.

Additional parameters and results were as follows: HPLC: Ret. Time: 5.6min; Assay >98% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (01% TFA);Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1441.7 [M-2H]/2, 960.9 [M-3H]/3. CE analysis(counter-ion % wt./wt.): TFA estimated to be 0.3%; acetate 1.1%.

For compound (26), following the same procedure for compound (23), 300mg of the peptide TFA salt (25) in 80 mL of water was loaded at 17mL/min. to a 150 mL of AG MP-50 column, which was washed with H₂O toconductivity of 1 μs/cm. The column was then washed with H₂O again afterloading, and the same step gradient of aqueous NH₄OAc into H₂O asemployed for the ion exchange of compound (23) was applied.Lyophilization of the combined fractions to a constant weight afforded200 mg of the acetate (26) as a white fluffy solid. The yield was 66.7%.

Additional parameters and results were as follows: HPLC: Ret. Time: 5.6min; Assay 97.0% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (01% TFA);Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1336.9 [M-2H]/2, 890.8 [M-3H]/3; CE analysis(counter-ion % wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; ICanalysis (F %): 0.26.

For the dimer peptide (27) acetate salt, similar to the procedure forcompound (23), an AG MP-50 column (100 mL wet volume) was washed with30% CH₃CN/H₂O until the conductivity was below 1 μs/cm. Compound (27) asthe TFA salt, (120 mg in 80 mL of 30% of CH₃CN/H₂O) was loaded onto thecolumn and the column was washed with the same eluent until theconductivity was stable at 1 μs/cm. A step gradient of NH₄OAc 30% ofCH₃CN/H₂O into 30% of CH₃CN/H₂O was run as for compound (23) and thecompound was eluted at ca 600 mM NH₄OAc. The combined fractions werelyophilized and then relyophilized several times until the materialdisplayed a constant weight to provide 104 mg of the pure material (27)as an acetate salt. The yield was 86.7%.

Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (0.1% TFA);Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5;CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.2%; acetate0.15%.

Preparation and Purification of the Dimer Peptide (27) Acetate Salt fromCompound (23) and Compound (26)

To a solution of disuccinimidyl glutarate (18 mg, 0.055 mmol) inanhydrous DMF (0.1 mL) was added a solution of compound (23) (61 mg,0.021 mmol) in 0.2 mL of anhydrous DMF dropwise (pH 8, neutralized byDIEA). The clear solution was stirred at RT for 0.5 h. HPLC and MSshowed the completion of the reaction. Solvent was removed in vacuo andEtOAc (8 mL) was added to precipitate the intermediate (24). The mixturewas centrifuged and decanted to remove excess glutarate. This EtOAcwashing was repeated 3 more times and the resulting solid was driedusing a stream of dry nitrogen. It was then dissolved in 0.3 mL ofanhydrous DMF. Compound (26), (56 mg, 0.021 mmol) was added and the pHof the solution was adjusted to 8 by addition of DIEA. The solution wasstirred for 16 h at room temperature after which by HPLC and MS analysisindicated completion of the reaction. A 30 μL aliquot of NH₂NH₂ wasadded and the mixture was stirred for 5 min to cleave the ivDde group.The reaction mixture was analyzed by HPLC and MS, which indicatedcomplete removal of the ivDde group.

Before purification of the dimer peptide (27) acetate, caution was takento carefully wash the whole preparative HPLC system including the columnwith TFA-free eluents, CH₃CN/H₂O/10 mM NH₄OAc. The crude reactionmixture was then applied to a reverse phase C-18 preparative column(Atlantis C-18, 5 μm particle, 100 Å pore, 30×150 mm, flow rate 30mL/min), pre-equilibrated with 15% B (A: 10 mM NH₄OAc in H₂O; B: 10 mMNH₄OAc in CH₃CN/H₂O, 9/1, v/v). The column was washed with the sameeluent until the DMF plug was eluted. The eluent composition wasincreased to 25% B over 2 min. and then ramped to 65% B over 40 min. Thefractions were analyzed on an analytical reverse phase C-18 column(Waters MS C-18, 4.6×50 mm, 5 μm particle, 100 Å pore, flow rate 3mL/min) and the product-containing fractions of >95% purity were pooledand freeze-dried to afford 25 mg of the dimer peptide (27) as itsacetate salt as a fluffy white solid. The yield was 21.8%.

Additional parameters and results were as follows: HPLC: Ret. time: 5.2min; Assay >99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μparticle, 120 Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN (0.1% TFA);Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min;Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES;Mode: Negative ion; [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CEanalysis (counter-ion % wt./wt.): TFA estimated to be less than 0.2%;acetate 1.1%.

Example 7 FIG. 7 Preparation of Dimer Peptide-Phospholipid ConjugatesHaving Low TFA Levels Via Ion Exchange Resin

Preparation and Purification of the Phospholipid Peptide Conjugate (21)as its Acetate Salt from Dimer Peptide (27) Acetate Salt

To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3 μmol) inanhydrous DMF (0.1 mL) was added a solution of neutralized dimer peptide(27) acetate salt, (15 mg, 2.75 μmol) in anhydrous DMF (0.2 mL),dropwise. The reaction solution was stirred at RT for 0.5 h. HPLCanalysis with a Waters Xterra C-18 column and MS showed the completionof the reaction. The solvent was evaporated and EtOAc (8 mL) was addedto precipitate the intermediate (28). The vessel containing theprecipitated intermediate (28) was centrifuged and the liquid layer wasdecanted. This procedure was repeated 3 times to remove the excess ofDSG. The solid was dried with a stream of dry nitrogen and thendissolved in 0.3 mL of anhydrous DMF. DSPE-PEG2000-NH₂ ammonium salt(29) (6.5 mg, 2.33 μmol) was added in solid form and the pH of themixture was adjusted to (28). The reaction mixture was stirred at RT for16 h. The mixture was analyzed by MS and HPLC with a Zorbax 300 SB-C3column and this indicated that the reaction was complete.

To minimize the potential contamination of the product with TFA, thecrude reaction mixture was purified by preparative HPLC equipped using anew Zorbax 300SB-C3 column (21.2×150 mm, 5μ particle) which had neverbeen exposed to TFA. The HPLC system was pre-washed by CH₃CN/H₂O/NH₄OAcextensively to remove traces of TFA. The reaction mixture was loadedonto the column which was pre-equilibrated with 20% B (A: 10 mM NH₄OAcin H₂O; B: 10 mM NH₄OAc in CH₃CN/H₂O, 9/1 v/v) at a flow rate of 30mL/min. The column was eluted at 30 mL/min with the same eluent untilthe plug of DMF was eluted. The eluent composition was then increased to40% B over 3 min and then ramped to 90% B over 50 min. The collectedfractions were analyzed on an analytical reverse phase C-3 column(Zorbax 300SB-C3, 3×150 mm, 3.5 μm particle, 300 Å pore, flow rate: 0.5mL/min), where detection was accomplished using UV at 220 nm and anevaporative light scattering detector (ELSD). The fractions containingthe pure product were pooled and lyophilized. A 6.5 mg portion of thefinal product (21) acetate salt was obtained. The yield was 33.0%.

Additional parameters and results were as follows: HPLC: Ret. Time: 13.3min; Assay >99% (area %); Column: Zorbax 300SB-C3, 3×150 mm, 3.5 μm, 300Å pore; Eluent: A: H₂O (0.1% TFA), B: CH₃CN/MeOH 1/1 (01% TFA); Elution:Initial condition: 60% B, linear gradient 60-90% B over 3 min; Flowrate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis(counter-ion % wt./wt.): % wt. TFA: 0.3%; % wt acetate 0.4%.

Example 8 FIG. 8

Preparation of Dimeric Conjugate Having Low TFA Levels Via SequentialPurification Using Zorbax C-3 RP Preparative HPLC and Sephadex G-25 GelPermeation Chromatography

Materials used and conditions for the analytical HPLC system include thefollowing: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle;Eluent A: H₂O (HPLC Grade with 0.1% TFA by volume); Eluent B: CH₃CN(0.1% TFA by volume). Elution: Initial condition: 50% B then a lineargradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate:0.5 mL/min; Detection: UV at 220 nm. Ret. time: (Compound (21)): 6.77min, Rt (lyso): 4.06 min.

Preparative HPLC using Preparative Zorbax C-3 Column to Remove theLyso-Compound from (21)

The crude compound was loaded at a concentration of 30% eluent B.Materials used and conditions include: Conditions: Column: Waters Zorbax300SB C-3; 21.2 mm i.d.×150 mm; 3.5 μm particle; Eluents: Eluent A:H₂O(HPLC Grade with 10 mM NH₄OAc); Eluent B: CH₃CN/H₂O, 9/1 (finalNH₄OAc concentration: 10 mM).

The composition of the eluent was then changed to 45% B over 2 min, thenthe column was eluted with a linear gradient of 45-100% B over 40 min;Flow rate: 30 mL/min; Detection: UV at 220 nm.

The crude compound (100 mg) was dissolved in 25 mL of a solution of 30%B. The preparative HPLC system was equilibrated at 30% B. The compoundwas loaded on to the Zorbax C-3 column. The mobile phase composition wasramped to 45% B over 2 min. A linear gradient from 45-100% B over 40 minwas used for the elution of (21). The product eluted between 26.5-33min.

The fractions that contained (21) were combined and lyophilized to givea white fluffy solid. This was dissolved in water-acetonitrile, thenlyophilized again. This provided 70 mg product devoid of thelyso-compound. The recovery was about 70%. After chromatography wascompleted, the system was washed with 95% B for 15 min at a flow rate of30 mL/min. The column was then washed with CH₃CN/H₂O (50/50, without TFAor buffer) for 30 min at a flow rate of 15 mL/min. The column was thenstored at room temperature for future use. Analytical HPLC confirmed theabsence of the lyso-compound in the isolated material. Further analysisconfirmed that no lyso-compound formed after 5 days at room temperature.The material still contained significant amounts (4.2 wt %) of TFA.

Removal of TFA from (21) by Gel Permeation Chromatography on SephadexG-25

A Sephadex G-25 column (100 g resin, bead size 20-80 μm, total gelvolume ˜500 mL, column height: 27 cm) was equilibrated with 4 L of 50 mMammonium bicarbonate. Then (21) (70 mg) was dissolved in 30 mL (finalvolume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. Thesolution was filtered and then loaded on to the Sephadex G-25 column.The column was eluted with 50 mM ammonium bicarbonate buffer withcollection of 10 mL fractions. The collected fractions were monitored byanalytical HPLC (UV detection at 220 nm). The results are provided inTable 4 below.

TABLE 4 Compound present (by HPLC analysis Fraction # Volume (mL) offraction) 1 10 No 3 10 No 6 10 No 9 10 No 12 10 No 15 10 No 18 10 No 1910 No 20 10 Yes 21 10 Yes 24 10 Yes 27 10 Yes 28 10 Yes 29 10 No

Fractions 20-28 were pooled and lyophilized. The lyophilized materialobtained was re-dissolved in a small volume of water and the solutionwas frozen and lyophilized to remove residual amounts of ammoniumbicarbonate. The final weight of the desired material was 58 mg. Therecovery was 83%.

To ascertain the effective removal of TFA, the sample was subjected toCE analysis for TFA and acetate ions. The TFA is clearly present in thestarting material (4.2%) according to the previous assay, while it ishardly detected (0.2%) after the gel permeation procedure. No acetateion was detected.

Analytical Data for (21) Obtained by Serial Zorbax C-3 Preparative HPLCand Sephadex G-25 gel Permeation Chromatography

Materials used and conditions for collecting analytical data include:Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt); Mass Spectrum:Method: MALDI-TOF; Mode: Positive Ion; Average molecular weight detectedwas 8461 the typical PEG2000 mass distribution curve was observed. HPLC:System A: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle;Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B:Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution:linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flowrate: 0.5 mL/min; Detection: UV at 220 nm. Ret time: 6.77 min; Area %:99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μmparticle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); EluentB: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution:linear gradient of 50-90% B over 3 min then ramp to 100% B over 12 min.Flow rate: 0.5 mL/min; Detection: LSD; Ret: time: 13.98 min. Area %:99.3%.

Table 5 below provides definitions for the abbreviations used and thesources of materials referred to in Examples 9-12.

TABLE 5 DSPA.Na (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphosphatidic acid, sodium salt DPPG.Na (Genzyme) IUPAC:1,2-Dipalmitoyl-sn-glycero-3- phosphoglycerol, sodium salt DPPE(Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine DSPCDistearoyl-glycero-phosphatidylcholine (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3-phosphocholine DSPG.Na (Genzyme) IUPAC:1,2-Distearoyl-sn-glycero-3- phosphoglycerol, sodium salt DSPE-PEG1000Distearoyl-glycero-phosphoethanolamine-N- methoxy(polyethyleneglycol)1000 (Avanti Polar) DSPE-PEG2000Distearoyl-glycero-phosphoethanolamine-N- methoxy(polyethyleneglycol)2000 (Avanti Polar) Stearate* Sodium Stearate (Fluka) PEG4000(polyethylene glycol) MW 4000 (Fluka) Mannitol (Fluka) *the acid form,i.e., stearic acid, can also be used in any of the microbubblepreparations herein.

Example 9 Preparation of Targeted Microbubbles with DSPC/DPPG EnvelopeExample 9A

383 mg of a mixture of DSPC/DPPG/and the dimeric peptide phospholipidconjugate (11) shown in FIG. 5 (molar ratio 49.75/49.75/0.5,corresponding to 187.1, 176.4 and 19.8 mg of the three components,respectively) and PEG-4000 (22.6 g) were solubilized in 120 g of t-butylalcohol at 60° C., in a water bath. The solution was filled in vialswith 0.8 mL of solution each. The samples were frozen at −45° C. andlyophilized. The air in the headspace was replaced with a mixture ofC₄F₁₀/Nitrogen (50/50) and vials capped and crimped. The lyophilizedsamples were reconstituted with 5 mL of H₂O per vial.

Example 9B

Example 9A was repeated using a mixture of DSPC/DPPG/and the monomericpeptide phospholipid conjugate (31) shown in FIG. 10 (molar ratio49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg of the threecomponents, respectively).

Example 10 Preparation of Targeted Microbubbles with DPPE/DPPG EnvelopeExample 10A

An aqueous suspension of DSPE-PEG1000 (0.43 mg-0.24 μmole) and themonomeric peptide phospholipid conjugate (31) shown in FIG. 10 (3.0mg-0.5 μmole) was prepared in 500 μL of distilled water at 60° C. toobtain a micellar suspension.

Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) weredispersed in a solution of mannitol 10% in distilled water (20 mL) at70° C. for 20 minutes. The dispersion was then cooled to roomtemperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueousphase using a high speed homogenizer (Polytron PT3000, probe diameter of3 cm) for 1 minute at 10500 rpm to obtain an emulsion.

The micellar suspension was added to the emulsion and the resultingmixture was heated at 60° C. for 1 hour under stirring. After cooling toroom temperature (1 hour), the obtained emulsion was divided in 4 mLfractions in 50 mL round bottom flasks. The emulsion was frozen at −45°C. for 5 minutes and freeze-dried at 0.2 mBar for 24 hours (Freeze-DrierChrist Beta 1-8K).

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C4F10/nitrogen (50/50 by volume). The lyophilized product wasthen dispersed in a volume of water twice the initial one by gentle handshaking.

Example 10B

An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.27 μmole) and dimericpeptidephospholipid conjugate (11) shown in FIG. 5 (5.3 mg-0.63 μmole)was prepared in 500 L of distilled water at 60° C. to obtain a micellarsuspension.

Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) weredispersed in a solution of PEG4000 10% in distilled water (20 mL) at 70°C. for 20 minutes. The dispersion was then cooled to room temperature.Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using ahigh speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1minute at 10000 rpm to obtain an emulsion.

The micellar suspension was added to the emulsion and the resultingmixture was heated at 80° C. for 1 hour under stirring. After cooling toroom temperature (1 hour), the obtained emulsion was washed once bycentrifugation (200 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of phospholipid. The separated pellet (containing emulsifiedmicrodroplets of solvent) was recovered and re-suspended with theinitial volume of a 10% PEG4000 aqueous solution.

The obtained emulsion was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mBar for 12 hours with a final dryingstep at 30° C. and 0.1 mBar for 7 hours. Vials were exposed to anatmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. Thelyophilized product was redispersed in a volume of water twice theinitial one by gentle hand shaking.

Example 11 Preparation of Targeted Microbubbles with DSPC/DSPA EnvelopeExample 11A

An aqueous suspension of DSPE-PEG1000 (2.5 mg-1.4 μmole) and dimericpeptide conjugate (11) shown in FIG. 5 (7.0 mg-0.84 μmole) was preparedin 1 mL of distilled water at 60° C. to obtain a micellar suspension.

Separately, DSPC (16.3 mg-20.6 μmoles) and DSPA (3.7 mg-5.15 μmoles)were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase wasadded to a PEG4000 10% solution in water (20 mL) using a high speedhomogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 800rpm, to obtain an emulsion.

The micellar suspension was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended intwice the initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to anatmosphere containing C₄F₁₀/Nitrogen (35/65 by volume) and sealed. Thelyophilized product was then dispersed in a volume of water twice theinitial one by gentle hand shaking.

Example 11B

Example 11A was repeated, but using 0.7 mg of DSPE-PEG2000 (0.26 μmoles)and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in FIG.2 (0.26 μmole) to prepare the micellar suspension.

Example 11C

DSPC (16.3 mg-20.6 μmoles), DSPA (3.7 mg-5.15 μmoles) and monomericpeptide phospholipid conjugate (1) shown in FIG. 1 (1.6 mg-0.26 μmole)were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase wasemulsified in a PEG4000 10% aqueous phase (20 mL) using a high speedhomogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at8000 rpm to obtain an emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling to room temperature (1 hour), the obtained emulsion wasdiluted with 20 ml of a PEG4000 10% aqueous solution.

The emulsion was sampled into DIN8R vials (1 mL/vial). Then vials werecooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-driedat −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C.and 0.1 mBar for 7 hours. Vials were exposed to an atmosphere containingC4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product wasredispersed in a volume of water twice the initial one by gentle handshaking.

Example 12 Preparation of Targeted Microbubbles with DSPC/StearateEnvelope Example 12A

An aqueous suspension of DSPE-PEG2000 (2.5 mg-0.9 μmoles) and thedimeric phospholipid conjugate (11) shown in FIG. 5 (2.5 mg-0.3 μmoles)was prepared in 660 μL of distilled water at 60° C. to obtain themicellar suspension.

Separately, DSPC (18.2 mg-23.1 μmoles) and stearate (1.8 mg-5.8 μmoles)were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase wasadded to a PEG4000 10% solution in water (20 mL) using a high speedhomogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at9000 rpm, to obtain an emulsion.

The micellar solution was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended withtwice the initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DIN8R vials (1 mL/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to anatmosphere containing C₄F₁₀/Nitrogen (35/65 by volume) and sealed. Thelyophilized product was dispersed in a volume of water twice the initialone by gentle hand shaking.

Example 12B

Example 12A was repeated by replacing the dimeric peptide phospholipidconjugate (11) shown in FIG. 5 with the same relative molar amount ofthe monomeric peptide phospholipid conjugate (1) shown in FIG. 2.

Example 12C

Example 11C was repeated with DSPC (18.2 mg-23.1 μmoles), sodiumstearate (1.8 mg-5.8 μmoles) and the dimeric peptide phospholipidconjugate (11) shown in FIG. 5 (2.2 mg-0.26 μmole). The agitation speedfor emulsification was fixed to 9000 rpm. After cooling to roomtemperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended intwice the initial volume of a 10% PEG 4000 aqueous solution.

Example 13 Static Binding Test on KDR-Transfected Cells

Plasmid Production and Purification

Full-length KDR was cloned into the pcDNA6 vector and the plasmid wasamplified in competent DH5α E. coli. Plasmid amplification andpurification was performed using E. coli JM 109 and a kit from Quiagen.

Transfection of 293H Cells on Thermanox® Coverslips

Cells were grown on poly-D-lysine-coated Thermanox® circular coverslipsin 24-well plate. Transfection was done as recommended in thelipofectamine 2000 protocol (Invitrogen, cat#11668-019) using 1 μg ofDNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2) in 0.1 mL. Transfection wasdone in serum-free media, the transfection reagent mix was removed fromcells after 2 hours and replaced with regular serum-containing medium.Some of the cell-coated coverslips were mock-transfected (with no DNA).The next day, expression of the KDR receptor was assessed byimmunocytochemistry and the binding assay was performed.

Bubble Binding Assay

The transfected cells were incubated with KDR-targeted microbubblesresuspended in 50% human plasma in PBS. For the incubation with thetransfected cells a small plastic cap was filled with a suspensioncontaining a 1.3×10⁸ bubbles and the cap was covered with an invertedThermanox® coverslip so as to put the transfected cells in contact withthe targeted microbubbles. After 30 min of incubation at RT, thecoverslip was lifted with tweezers, rinsed three times in PBS andexamined under a microscope to assess binding of the targetedmicrobubbles.

Determination of the % of Surface Covered by Microbubbles

Images were acquired with a digital camera DC300F (Leica) and thepercent of surface covered by bound microbubbles in the imaged area wasdetermined using the software QWin version 3.1 (Leica Microsystem AG,Basel, Switzerland). Pictures were taken of each Thermanox®coverslip.For each preparation of Examples 9 and 10, the binding assay wasrepeated a minimum of two times thus obtaining an average value of thesurface covered. In the following Tables 6 and 7, the binding activityof the microbubbles prepared according to Examples 9 and 10 above arerecorded.

As indicated by the Tables, the same peptide may show different bindingactivities when included (as a lipopeptide) in different phospholipidformulations forming the stabilizing envelope of the microbubble.Microbubbles containing KDR binding lipopeptides of the invention bindspecifically to KDR-expressing cells while they did not bind appreciablyto mock transfected cells.

Example 14 Dynamic Binding test on rh VEGF-R2/Fc Chimeric Protein

Preparation of Fc-VEGF-R2-Coated Coverslips

Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, Pa., USA)were coated with recombinant human VEGF-R2/Fc Chimeric protein (R&DSystems) according the following methodology.

A surface of dimensions 14×25 mm was delimited on the glass coverslipusing a special marker (Dako Pen) and 400 μL of Fc-VEGF-R2 solution at 4μg/mL in PBS was deposited on this surface. After an overnightincubation at 4° C., the solution was aspirated, replaced by 0.5 mL of asolution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3hours at RT. Then the coverslip was washed three times with 5 ml ofPBS-0.05% Tween 80.

Binding Assay

Binding studies of targeted bubbles were carried out using aparallel-plate flow chamber (FCS2, Bioptech Inc., Butler, Pa., USA) witha chamber gasket of 0.25 mm in thickness, with a customized adapter forupside-down chamber inversion. The coated coverslip was inserted as aplate of the flow chamber. Microbubbles (5×10⁶ bubbles/mL in 50% humanplasma in PBS) were drawn through the flow chamber using an adjustableinfusion pump (Auto Syringe® AS50 Infusion Pump, Baxter, Deerfield,Ill., USA) with a 60 mL syringe (Terumo). The pump flow rate wasadjusted to 1 mL/min to obtain the desired shear rate of about 114 s⁻¹.After 10 minutes, the flow was stopped and pictures were taken randomlyat different positions on the coverslip (on areas of about 0.025 mm²)using a 40× objective and a CCD monochrome camera (F-View II, SoftImaging Systems, Germany) connected to an inverted Olympus IX 50microscope.

The number of microbubbles on each picture was determined, averaged withrespect to the total number of pictures and the obtained value was thendivided by ten (to obtain the “slope”, i.e. the average amount of boundmicrobubbles per minute).

For each preparation of Examples 11 and 12, the binding assay wasrepeated four times thus obtaining an average value of the slope.

The slope represents the bubble binding rate on the target substrate.For instance, a slope value of 8 indicates that an average of eighty(80) microbubbles was bound on the coated coverslip in ten minutes. Ahigher slope indicates a better capacity of bubbles to bind to thetarget under flow conditions.

In the following tables 8 and 9, the binding activity of themicrobubbles prepared according to Examples 11 and 12 above wereillustrated.

As inferable from the tables, the same peptide may show differentbinding activities when included (as a peptide-phospholipid conjugate orlipopeptide) in different phospholipid formulations forming thestabilizing envelope of the microbubble.

TABLE 6 Example KDR Mock KDR-Mock 9A 28.6% 0.4% 28.3% 9B 28.0% 0.3%27.7%

TABLE 7 Example KDR Mock KDR-Mock 10A 23.6% 0.2% 23.5% 10B 28.0% 0.0%28.0%

TABLE 8 Example Slope 11A 8.2 11B 8.1 11C 5.8

TABLE 9 Example Slope 12A 9.0 12B 8.0 12C 7.8

Example 15 In Vivo Evaluation of Ultrasound Contrast Agents Targeted toKDR

The ability of ultrasound contrast agents containing KDR bindinglipopeptides of the invention to bind to KDR-expressing tissue in vivowas assessed using a known model of angiogenesis: the rabbit VX2 tumormodel.

A known model of angiogenic tissue was used to examine the ability ofthe KDR-targeted ultrasound microbubbles to localize to and provide animage of angiogenic tissue. The VX2 rabbit carcinoma was seriallyimplanted in the dorsal muscle of New Zealand rabbits (Charles RiverLaboratories, France) weighting 2.5/3 kg.

Preparation of Tumor Homogenate

Tumor was surgically removed, placed into McCoy's culture mediumcontaining 10% fetal calf serum, antibiotics, 1.5 mM Glutamax 1 and cutinto small pieces that were rinsed to remove blood and debris. Thentumor pieces (3 to 5 cm³) were placed in a 50 ml Falcon tube containing5 mL of complete medium. The tumor tissue was ground (Polytron) until nomore solid pieces were visible. The murky fluid was centrifuged for 5minutes at 300 g and the supernatant discarded. Seven mL of fresh mediumwas added per 5 mL of pellet.

Tumor Implantation

Rabbits received first 0.3 mL of Vetranquil (Acepromazine, Sanofi,injected intramuscularly) and were then anesthetized with anintramuscular injection of Ketaminol®5/Xylazine (Veterinaria AG/Sigma)mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters of VX2 tumorhomogenate was injected intramuscularly. Fifteen days after implantationof VX2 tumors, animals were anesthetized again with the same mixture,plus subcutaneous injection of 50% Urethane (2 mL/kg, s.c.) (Sigma) forimaging experiments.

In Vivo Ultrasound Imaging

VX2 tumor imaging was performed using an ultrasound imaging system ATLHDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulseinversion at high acoustic power (MI=0.9) was used to evaluateaccumulation of targeted microbubbles on the KDR receptor expressed onthe endothelium of neovessels. The linear probe was fixed on the skindirectly over the implanted tumors.

After bubble injection (0.1 μL/kg of gas) using the preparations ofeither Example 16 or Example 17, insonation was stopped allowing bubblesto accumulate for 25 minutes. Then, insonation was reactivated at highacoustic power (MI 0.9) destroying all the bubbles present in the tumor.The amount of free circulating bubbles was then assessed by recordingthe signal obtained after 20 sec accumulation without insonation.

Video frames from VX2 tumor imaging experiments were captured withvideo-capture and analysed with Image-Pro Plus 2.0 software. The imagerepresenting free circulating bubbles was subtracted from the imageobtained at 25 min, to provide an image representing bound bubbles.Referring to FIG. 11 (which shows the results with the preparation ofExample 16) and FIG. 12 (which shows the results with the preparation ofExample 17), FIGS. 11A and 12A show an image before bubble injection(baseline); FIGS. 11B and 12B show retention of bubble contrast in thetumor 25 minutes post injection; and FIGS. 11C and 12C show the resultobtained after subtraction of the baseline and free circulating bubblesand represent bound microbubbles containing KDR lipopeptides accordingto the present invention. Examples 15-17 and FIGS. 11 and 12 confirmthat ultrasound contrast agents bearing such KDR binding moietieslocalize to KDR expressing (and thus angiogenic) tissue in animalmodels.

Example 16

Example 12A was repeated by replacing DSPE-PEG2000 with DSPE-PEG1000(2.7 mg, 1.54 μmol) and using 2.5 mg (0.31 μmol) of dimeric peptidephospholipid conjugate (11) shown in FIG. 5.

Example 17

Example 16 was repeated by replacing the dimeric peptide phospholipidconjugate with the same molar amount of monomeric phospholipid conjugate(1) shown in FIG. 2.

1. An ultrasound contrast agent composition comprising one or morepeptide monomers selected from the group consisting of:Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6); andAc-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7).
 2. The compositionof claim 1, wherein the contrast agent comprises a gas filledmicrovesicle.
 3. The composition of claim 2, wherein said gas comprisesa fluorinated gas.
 4. The composition of claim 2, further comprising aphospholipid.
 5. The composition of claim 4 further comprising two ormore components selected from the group consisting of: DSPC, DPPG, DPPA,DSPA, DPPE, DPPG, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearicacid.
 6. The composition of claim 5, wherein the gas the gas comprisesC₃F₈, C₄F₁₀ or SF₆, optionally in admixture with air, nitrogen, oxygenor carbon dioxide.
 7. The composition of claim 5, further comprising acomponent selected from the group consisting of: sugars, polysaccharidesand polyols.
 8. The composition of claim 7, wherein said component isselected from the group consisting of: mannitol, dextran andpolyethyleneglycol.
 9. A peptide-phospholipid conjugate comprising apeptide monomer selected from the group consisting ofAc-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2 (SEQ. ID NO. 6); andAc-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2 (SEQ. ID NO. 7).
 10. Thepeptide-phospholipid conjugate of claim 9, wherein the phospholipid isselected from the group consisting of: phosphatidylethanolamines andmodified phosphatidylethanolamines.
 11. The peptide-phospholipidconjugate of claim 10, wherein the phospholipid is DSPE-PEG2000.
 12. Anultrasound contrast agent composition comprising a conjugate of claim 9.13. The composition of claim 12, wherein the contrast agent comprises agas-filled microvesicle.
 14. The composition of claim 13, wherein thegas filled microvesicle comprises a phospholipid.
 15. The composition ofclaim 14, wherein the gas-filled microvesicle further comprises two ormore components selected form the group consisting of: DSPC, DPPG, DPPA,DSPA, DPPE, DPPG, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearicacid.
 16. The composition of claim 15, wherein the gas-filledmicrovesicle comprises DSPE-PEG2000.
 17. The composition of claim 15,wherein the gas-filled microvesicle comprises DSPG and DPPG.
 18. Thecomposition of claim 15, wherein the gas filled microvesicle comprisesDPPE and DPPG.
 19. The composition of claim 15, further comprising acomponent selected from the group consisting of: sugars, polysaccharidesand polyols.
 20. The composition of claim 19, wherein said component isselected from the group consisting of: mannitol, dextran andpolyethyleneglycol.
 21. The composition of claim 13, wherein said gascomprises a fluorinated gas.
 22. The composition of claim 21, whereinthe gas comprises C₃F₈, C₄F₁₀ or SF₆, optionally in admixture with air,nitrogen, oxygen or carbon dioxide.
 23. A method for imagingKDR-containing tissue in a mammal comprising administering an effectiveamount of a composition of any one of claims 5 or 15 to the mammal andimaging the mammal.
 24. A method for detecting or imaging angiogenicprocesses in a mammal comprising administering an effective amount of acomposition of any one of claims 5 or 15 to the mammal and imaging themammal.
 25. A method for detecting or imaging tumor tissue containingKDR in a mammal comprising administering an effective amount of acomposition of any one of claims 5 or 15 to the mammal and imaging themammal.
 26. A KDR-binding peptide monomer selected from the groupconsisting of: (SEQ. ID NO. 6) Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH2; and(SEQ. ID NO. 7) Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH2.